EP1804584A2 - Verfahren und zusammensetzungen im zusammenhang mit der verabreichung eines ganoderma-lucidum-extrakts - Google Patents

Verfahren und zusammensetzungen im zusammenhang mit der verabreichung eines ganoderma-lucidum-extrakts

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Publication number
EP1804584A2
EP1804584A2 EP05810080A EP05810080A EP1804584A2 EP 1804584 A2 EP1804584 A2 EP 1804584A2 EP 05810080 A EP05810080 A EP 05810080A EP 05810080 A EP05810080 A EP 05810080A EP 1804584 A2 EP1804584 A2 EP 1804584A2
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EP
European Patent Office
Prior art keywords
fucose
cell
containing glycoprotein
glycoprotein fraction
effective amount
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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EP05810080A
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English (en)
French (fr)
Other versions
EP1804584A4 (de
Inventor
Chi-Huey Wong
Hsien-Yeh Hsu
Kuo-Feng Hua
Chun-Hung Lin
Jason Hsu
Shui-Tein Chen
Kuo-I Lin
Wen-Bin Yang
John Yu
Alice Yu
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Academia Sinica
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Academia Sinica
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Publication of EP1804584A2 publication Critical patent/EP1804584A2/de
Publication of EP1804584A4 publication Critical patent/EP1804584A4/de
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/074—Ganoderma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • Field of the Disclosure refers to the field of immunology and cellular biology.
  • Ganoderma species (a group of medical fungus) is known in the art.
  • Ganoderma lucidum (Reishi or Ling-Zhi) has been used as traditional Chinese medicine (TCM) for promoting good health, perpetual youth, and longevity and in particular extract of Reishi were used as anti-tumor and immuno-modulating agent.
  • Reishi is also known to exhibit liver protective, hypoglycemic and platelet aggregation-inhibiting activities.
  • Chemical composition of the extract includes polysaccharides having the structure shown in Fig. 1. Although the receptor complement receptor type three (CR3) has been shown to bind the ⁇ -glucan polysaccharides, the receptor, and carbohydrate epitope responsible for the anti-tumor activity remain to be established.
  • CR3 receptor complement receptor type three
  • a fucose-containing glycoprotein fraction from an extract of Ganoderma Lucidum Reishi or Ling Zhi is disclosed, the fucose- containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues.
  • a method to mediate a biological event associated with activated expression of a cytokine is disclosed.
  • a method to mediate inimmunomodulating events associated with IL-1 IL6, IFN and/or TNF gene expression in a mammalian cell is disclosed.
  • the method comprises administering to the cell an effective amount of a fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues.
  • the immunomodulating events can comprise the secretion of lnterleukin-1 IL6 IFN and/or TNF, and/or the secrection precursor thereof and the expression of lnterleukin 1 converting enzyme.
  • the effective amount of the fucose-containing glycoprotein fraction can be an amount inducing an up-regulation of the secretion of interleukin-1 and/or precursor thereof and/or an amount inducing an up-regulation of the expression of lnterleukin-1 converting enzyme.
  • a method to stimulate the expression of an inflammatory cytokine in a mammalian cell comprises administering to the cell an effective amount of a fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues.
  • a method to modulate protein kinase pathways associated with inflammatory cytokine lnterleukin 1 in a mammalian cell comprises administering to the cell an effective amount of a fucose-containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues.
  • the effective amount of the fucose-containing glycoprotein fraction can be an amount activating PKC, MEK1 , PAK and/or MAPKs in the cell, the activated PKC, MEK1 , PAK and/or MAPKs leading to multiple cytokine production.
  • the effective amount of the fucose-containing glycoprotein fraction can be an amount activating a PKC/MEK1/ERK signal transduction pathway the activated pathway resulting in a transcriptional, posttranscriptional and posttranslational regulation of the lnterleukin 1 gene expression.
  • a method to induce a Toll-Like Receptor (TLR) mediated event in a mammalian cell comprises administering to the cell an effective amount of at least one of the above mentioned fucose-containing glycoprotein fraction.
  • TLR Toll-Like Receptor
  • the TLR mediated event can be the production of inflammatory cytokine and the inflammatory cytokine lnterleukin-1.
  • a method to modulate differentiation of a mononuclear cell comprising administering to the mononuclear cell an effective amount of a fucose-containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues.
  • the effective amount of the fucose-containing glycoprotein fraction can be an amount resulting in an increased CD14 + CD26 + expression in the cell and in a monocyte/macrophage differentiation of the cell.
  • the effective amount of the fucose-containing glycoprotein fraction can also be an amount resulting in an increased CD83 + CD1a + expression in the cell and in a dendritic cell differentiation of the cell.
  • the effective amount of the fucose-containing glycoprotein fraction can further be an amount resulting in an increased CD16 + CD56 + expression in the cell and in a NK cells differentiation of the cell.
  • a method to enhance cytotoxicity of an NK cell against an NK-sensitive tumor cell comprises administering to the NK cell an effective amount of a fucose-containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose- containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues.
  • the effective amount of the fucose-containing glycoprotein fraction can be an amount resulting in an increased cytotoxicity of the NK against the NK- sensitive tumor cells.
  • the NK cell can be a CD3 + CD56 + NKT cell or CD3 ' CD56 + NK cell.
  • a composition for mediating an immunomodulating event associated with IL-1 expression in a mammalian cell comprising an effective amount of a fucose- containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues, in a compatible vehicle carrier or auxiliary agent.
  • a composition for stimulating the expression of an inflammatory cytokine in a mammalian cell comprising an effective amount of a fucose-containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues, in a compatible vehicle, carrier, or auxiliary agent.
  • a composition for modulating protein kinase pathways associated with inflammatory cytokine lnterleukin 1 in a mammalian cell comprises an effective amount of a fucose- containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues, in a compatible, vehicle, carrier or auxiliary agent.
  • compositions for inducing a TLR mediated event in a mammalian cell comprising an effective amount of a fucose-containing glycoprotein fraction of an extract of
  • the TLR-mediated event can be the production of lnterleukin-1 in the cell.
  • a composition for modulating differentiation of a mononuclear cell comprises an effective amount of a fucose-containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues, in a compatible vehicle, carrier or auxiliary agent.
  • a composition for enhancing cytotoxicity of an NK cell against an NK-sensitive tumor cell comprises an effective amount of a fucose-containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues, in a compatible vehicle, carrier or auxiliary agent.
  • the NK cell is a CD3 + CD56 + NKT cell or CD3 " CD56 + NK cell.
  • a method to activate the expression of a cytokine in a mammalian cell comprises administering to the cell an effective amount of a fucose-containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues.
  • the cytokines are preferably selected from the group consisting of IL-1 , IL6, IL-12, IFN- ⁇ , TNF- ⁇ , GM-CSF, G-CSF and M-CSF.
  • a method to induce expression of Blimp 1 is in a mammalian cell, disclosed.
  • the method comprises administering to the cell an effective amount of the fucose-containing glycoprotein fraction.
  • a method to inhibit LPS induced nitric oxide production in macrophages comprises administering to the macrophage an effective amount of a fucose-containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose- containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues.
  • a method to activate spleen cell proliferation comprising administering to the mouse spleen cells an effective amount of a fucose-containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues.
  • a method to modify the proteome of spleen cells comprising administering to the mouse spleen cells an effective amount of a fucose-containing glycoprotein fraction of an extract of Ganoderma Lucidum Reishi, the fucose-containing glycoprotein fraction including at least one of a polysaccharide and a glycopeptide comprising fucose residues.
  • Fig. 1 shows a schematic representation of the backbone of Reishi polysaccharide fractions as known in the art
  • Fig. 2 shows a diagram illustrating the results of a gel filtration chromatography of Reishi extract performed using Sephacryl S-500 column; the x-axis shows the volume of the elute in ml_; the y axis shows the light absorbance of the elute at OD 625;
  • Fig. 3 shows a diagram illustrating a colorimetric assay (MTT) for Con A- stimulated cell proliferation of mouse spleen cells treated with crude Reishi extract; the x-axis shows the concentration of crude Reishi extract in ⁇ g/mL; the y-axis shows the index compared with the (-) control;
  • MTT colorimetric assay
  • Fig. 4 shows a diagram illustrating a colorimetric assay (MTT) for Con A- stimulated cell proliferation of mouse spleen cells treated with Fraction 3; the x- axis shows the concentration of crude Reishi extract in ⁇ g/mL; the y-axis shows the index compared with the (-) control; Fig.
  • lane 5 shows the results of gel electrophoresis of the RT-PCR experiments to detect the expression of IL-1 , in mouse spleen cells were incubated with fraction-3; lane 1 shows a molecular weight marker (50 bp ladder); lane 2 shows the expression of a house keeping gene (200 bp) in presence of Fraction 3; lane 3 shows IL-1 expression (152 bp) in presence of Fraction 3; lane 4 shows the expression of the house keeping gene in absence of Fraction 3; lane 5 shows IL-1 expression in absence of Fraction 3; fragments dimension is indicated on the left by arrows;
  • Fig. 6 shows the results of gel electrophoresis of the RT-PCR experiments to detect the expression of IL-2, in mouse spleen cells incubated with fraction-3; lane 1 shows a molecular weight marker (50 bp ladder); lane 2 shows the expression of a house keeping gene (200 bp) in presence of Fraction 3; lane 3 shows IL-2 expression (167 bp) in presence of Fraction 3; lane 4 shows the expression of the house keeping gene in absence of Fraction 3; lane 5 shows IL-2 expression in absence of Fraction 3; fragments dimension is indicated on the left by arrows; Fig.
  • lane 7 shows the results of gel electrophoresis of the RT-PCR experiments to detect the expression of IFN- ⁇ in mouse spleen cells incubated with fraction-3;
  • lane 1 shows a molecular weight marker (50 bp ladder);
  • lane 2 shows the expression of a house keeping gene (200 bp) in presence of Fraction 3;
  • lane 3 shows IFN- ⁇ expression (336 bp) in presence of Fraction 3;
  • lane 4 shows the expression of the house keeping gene in absence of Fraction 3;
  • lane 5 IFN-Y expression in absence of Fraction 3; fragments dimension is indicated on the left by arrows;
  • Fig. 8 shows the results of a proteomic analysis of mouse spleen cells in absence of treatment with Fraction 3;
  • Fig. 9 shows the results of a proteomic analysis of mouse spleen cells following treatment with Fraction 3;
  • Fig. 10 shows the disappearing spots from the proteomic analysis of Fig. 8 when compared with the proteomic analysis of Fig. 9
  • Fig. 1 1 shows the appearing spots in the proteomic analysis of Fig. 8 when compared with the proteomic analysis of Fig. 9;
  • Fig. 12 shows a chart illustrating a model to interpret the relationship between enzymatic treatment and bioactivity of Fraction 3;
  • Fig. 13 shows a gel electrophoresis of RT-PCR experiments showing the expression of 10 different cytokines following incubation for 6 h of mouse spleen cells with F3 at the concentration of 0.1 ppm (0.1 ppm), 1 ppm (1 ppm), 10 ppm
  • Fig. 14 shows a diagram reporting the results of an anion exchange chromatography carried out on a column of Diaion-WA30 (CP form, 40 * 3.5 cm) eluted with 0.2 and 0.8 M NaCI at a flow rate of 0.5 mL/min; the x axis shows the samples analyzed for light absorbace identified by the tube number; the y-axis shows the light absorbance at 480 nm; portions of the curves referred to Fractions F3G1 and F3G2, are identified in the diagram by the respective acronym;
  • Fig. 15 shows a diagram reporting the results of a chromatography carried out on a gel-filtration column TSK HW-75 (130 x 2.6 cm) eluted with double distilled water at a flow rate of 0.5 mL/min; the x axis shows the samples analyzed for light absorbance, the samples identified by the tube number; the y- axis shows the light absorbance at 480 nm; portions of the curves referred to fractions F3G2H1 (G2H1 ) and F3G2H2 (G2H2), are identified in the diagram by the respective acronym;
  • Fig. 16 shows a diagram illustrating the production in vitro of GM-CSF by mouse splenocytes (3.0 * 10 6 cells/mL) at different time intervals after treatment with 100 ⁇ g/mL of Reishi crude extract (WSR), F3 (F3), F3G1 (F3G1), and F3G2 (F3G2) as indicated in the figure;
  • WSR Reishi crude extract
  • F3 F3
  • F3G1 F3G1
  • F3G2 F3G2
  • Fig. 17 shows a diagram illustrating the production in vitro of IFN- ⁇ by mouse splenocytes (3.0 x 10 6 cells/mL) at different time intervals after treatment with 100 ⁇ g/mL of Reishi crude extract (WSR), F3 (F3), F3G1 (F3G1 ), and F3G2 (F3G2) as indicated in the figure;
  • WSR Reishi crude extract
  • F3 F3
  • F3G1 F3G1
  • F3G2 F3G2
  • Fig. 18 shows a table illustrating the expression in vitro of TNF- ⁇ by mouse splenocytes upon administration of either F3G2 or LPS at various concentrations as indicated; the first column shows the F3G2 or LPS concentrations (sample cone) in ⁇ g/mL; the second column shows the concentrations of TNF- ⁇ in pg/ml detected following F3G2 administration; the third column shows the concentrations of TNF- ⁇ in pg/ml detected following LPS administration; Fig.
  • FIG. 19 shows a three-dimensional chart illustrating the expression in vitro of TNF- ⁇ by mouse splenocytes upon administration of either F3G2 or LPS at various concentrations as indicated; the length axis shows the concentration of the F3G2 or LPS concentrations (sample cone) in ⁇ g/mL; the height axis shows the concentrations of TNF- ⁇ in pg/ml detected following F3G2 or LPS administration;
  • Fig. 20 shows the results of a Western Blot of whole cell lysates from HeNC2 and GG2EE cells treated for 6 h (+) with 25 ⁇ g/ml F3 (F3) and/or 1 ⁇ g/ml LPS (LPS) or not treated (-) as indicated; the detected expression of pro-IL-1 and actin (as an internal control) is indicated by arrows on the right;
  • Fig. 21 shows the results of a Western Blot of whole cell lysates from human primary macrophages pretreated for 30 min (+) with 10 ⁇ g/ml of mouse anti-human TLR4 mAb HTA125 (TLR4Ab) or 10 ⁇ g/ml of CR3 mAb (CR3Ab) or not pretreated (-) as indicated, the macrophages thereafter treated for 6 h (+) with 25 ⁇ g/ml of F3 (F3) or 1 ⁇ g/ml LPS (LPS) or not treated (-) as indicated; the detected expression of pro-IL-1 and actin (as an internal control) are indicated by arrows on the right;
  • Fig. 22 shows a diagram illustrating the F3-induced and LPS-induced ROS production by J774A.1 cells, upon preincubation of the J774A.1 cells with 2 ⁇ M CM-H 2 DCFDA for 30 min, followed by substitution with medium containing 25 ⁇ g/ml F3 (F3) and/or 1 ⁇ g/ml LPS (LPS) or no additional substance (DCPH only), as indicated, for additional incubation over a short period of time between 0 and 20 min; the x-axis shows the time of measurement expressed in minutes; the y-axis shows the relative fluorescence intensity of fluorophore CM-H 2 DCF; Fig.
  • FIG 23 shows a diagram reporting F3-induced and LPS-induced ROS production, upon preincubation of J774A.1 cells with 2 ⁇ M CM-H 2 DCFDA for 30 min, followed by substitution with medium containing 25 ⁇ g/ml F3 (F3) and/or 1 ⁇ g/ml LPS (LPS) or no additional substance (DPCOH only), as indicated, for additional incubation over a long period of time between 0 and 24 h; the x-axis shows the time of measurement expressed in hours; the y-axis shows the relative fluorescence intensity of fluorophore CM-H 2 DCF;
  • Fig. 24 shows the results of a Western Blot analysis with anti-IL-1 antiserum of whole cells lysates of J774A.1 cells pre-incubated with 10 ⁇ g/ml PMB for 30 min (PMB) or without PMB (blank), followed by treatment for 6h with
  • Fig. 25 shows the results of a Western Blot analysis with anti- phosphotyrosine mAb (anti-P-Tyr Ab 4G10) of human blood monocyte-derived macrophages, pre treated with 25 ⁇ g/ml F3 (F3), 1 ⁇ g/ml LPS (LPS), or medium (CTR) for different times expressed in minutes as indicated; sample 1 , untreated control; sample 2, 1 ⁇ g/ml LPS, 15 min; sample 3, 1 ⁇ g/ml LPS, 60 min; sample 4, 25 ⁇ g/ml F3, 15 min; sample 5, 25 ⁇ g/ml F3, 60 min; the molecular mass (kDa) are indicated by bars on the left side; expression of MAPKs confirmed specific Abs is indicated by bars on the right side; several additional phosphotyrosyl proteins F3 induced and not LPS-induced are indicated by an asterisk on the right side (*);
  • Fig. 26 shows a diagram illustrating the effect of 25 ⁇ g/ml F3 (F3), 1 ⁇ g/ml
  • LPS LPS
  • LY2940002/EORP LY2940002/EORP
  • Fig. 27 shows the results of a Western blot analysis with anti-IL-1 antiserum of whole cells lysates of J774A.1 cells (J774A.1 cells) treated for 6 h with 12.5 or 25 ⁇ g/ml F3 (F3 ⁇ g/ml) or 0.5 or 1 ⁇ g/ml LPS (LPS ⁇ g/ml) as indicated; the detected expression of pro-IL-1 and actin (as an internal control) is indicated by arrows on the right; Fig.
  • FIG. 28 shows the results of a Western blot analysis with anti-IL-1 antiserum of whole cells lysates of J774A.1 cells (J774A.1 cells) and human primary macrophages (Human primary macrophages) treated with F3 (F3), and specifically at 0 hours, (0), 3 hours (3), 6 hours (6), 9 hours (9), 12 hours (12), 24 hours (24) as indicated; the detected expression of pro-IL-1 and actin (as an internal control) are indicated by arrows on the right;
  • Fig. 29 shows the results of a Western blot analysis with anti-IL-1 antiserum of whole cells lysates of J774A.1 cells (J774A.1 cells) treated with F3 (F3) at 0 ⁇ g/ml (0), 0.3 ⁇ g/ml (0.3), 1.5 ⁇ g/ml (1.5), 3.1 ⁇ g/ml (3.1) 6.3 ⁇ g/ml (6.3) 12.5 ⁇ g/ml (12.5) 25 ⁇ g/ml (25) and 50 ⁇ g/ml (50) as indicated; the detected expression of pro-IL-1 and actin (as an internal control) are indicated by arrows on the right;
  • Fig. 30 shows results of RT-PCR analysis using probes specific for pro-IL- 1 of total RNA isolated from J774A.1 cells (J774A.1 cells) pre-treated with F3 (F3) at 0 hours, (0), 1 hour (1 ), 2hours (2), 4 hours (4), 6 hours (6), 8 hours (8), 12 hours (12), 18 hous (18) and 24 hours (24) as indicated; the detected expression of pro-IL-1 and GAPDH (as an internal control) are indicated by arrows on the right; presence or absence of enhanced expression and intensity of the enhanced expression is indicated on the lower portion of the figures (fold); Fig.
  • FIG. 31 shows a diagram illustrating a time-dependent F3-induced ICE activation of J774A.1 cells treated with F3 (F3) or with LPS (LPS); the x-axis shows the time of measurement espressed in hours; the y-axis ICE activity measured fluorometrically;
  • Fig. 32 shows the results of a Western Blot analysis with anti- diphosphorylated p44/42 of whole cells lysates of J7774A.1 (J7774A.1) pretreated with 25 ⁇ g/ml F3 (F3) at 0 minutes (0), 10 minutes (10), 20 minutes (200, 30 minutes (30), 60 minutes (60) and 120 minutes (120), as indicated; the detected expression of p-ERK, p-Elk and ERK is indicated by arrows on the right; Fig.
  • Fig. 34 shows the results of a Western Blot analysis with anti- diphosphorylated p38 mAb of whole cells lysates of J7774A.1 (J7774A.1) pretreated with 25 ⁇ g/ml F3 (F3) at 0 minutes (0), 10 minutes (10), 20 minutes (200, 30 minutes (30), 60 minutes (60) and 120 minutes (120), as indicated; the detected expression of p-p38, p-ATF-2 and p38 is indicated by arrows on the right;
  • Fig. 35 shows a diagram illustrating the quantification by Phosphorlmager using imageQuaNT software from Amersham Pharmacia Biotech (Sunnyvale, CA) of F3-stimulated phospho-ERK1/2 (p-ERK1/2), phospho-JNK1/2 (P- JNK1/2), and phospho-p38 (p-p38), as indicated, in J774A.1 cell samples tested as reported in Figs. 32 to 34 above; the x-axis shows the time of measurement expressed in minuted; the y-axis shows the relative fold;
  • Fig. 36 shows a diagram illustrating the quantification by Phosphorlmager using ImageQuaNT software from Amersham Pharmacia Biotech of F3- stimulated ERK1/2 (ERK) and p38 (p38) activity in J774A.1 cell samples tested as reported in Figs. 32 to 34 above; the x-axis shows the time of measurement expressed in minutes; the y-axis shows the relative MAPKs activity;
  • Fig. 37 shows the results of a Western blot analysis with anti-IL-1 antiserum of whole cells lysates of J774A.1 cells (J774A.1 cells) pre-incubated for 1 h with Ro-31-8220 (Ro-31-8220), 0 ⁇ M (0), 0.1 ⁇ M (0.1), 1 ⁇ M (1), 5 ⁇ M (5) or 10 ⁇ M (10), as indicated, followed by stimulation for additional 6 h (+) by 25 ⁇ g/ml F3 (F3) or no stimulation (-) as indicated; the detected expression of pro- IL-1 and actin (as an internal control) are indicated by arrows on the right;
  • Fig. 38 shows the results of a Western blot analysis with anti-IL-1 antiserum of whole cells lysates of J774A.1 cells (J774A.1 cells) pre-incubated for 1 h with PD98059 (PD98059) 0 ⁇ M (0), 5 ⁇ M (5), 25 ⁇ M (25), 50 ⁇ M (50) or 100 ⁇ M (100), as indicated, followed by stimulation for additional 6 h (+) by 25 ⁇ g/ml F3 (F3) or no stimulation (-) as indicated; the detected expression of pro- IL-1 and actin (as an internal control) are indicated by arrows on the right; Fig.
  • FIG. 39 shows the results of a Western blot analysis with anti-IL-1 antiserum of whole cells lysates of J774A.1 cells (J774A.1 cells) pre-incubated for 1 h with SP600125 (SP600125) 0 ⁇ M (0), 1 ⁇ M (1), 5 ⁇ M (5), 10 ⁇ M (10) or 20 ⁇ M (20), f followed by stimulation for additional 6 h (+) by 25 ⁇ g/ml F3 (F3) or no stimulation (-) as indicated; the detected expression of pro-IL-1 and actin (as an internal control) is indicated by arrows on the right;
  • Fig. 40 shows the results of a Western blot analysis with anti-IL-1 antiserum of whole cells lysates of J774A.1 cells (J774A.1 cells) pre-incubated for 1 hour with SB203580 (SB203580) 0 ⁇ M (0), 0.1 ⁇ M (0.1), 0.5 ⁇ M (0.5), 1 ⁇ M (1) or 2 ⁇ M (2), as indicated, followed by stimulation for additional 6 h (+) by 25 ⁇ g/ml F3 (F3) or no stimulation (-) as indicated; the detected expression of pro- IL-1 and actin (as an internal control) is indicated by arrows on the right;
  • Fig. 41 shows the results of a Western blot analysis with anti- diphosphorylated JNK1/2 and anti-diphosphorylated p38 mAb of whole cells lysates of J774A.1 cells (J774A.1 cells) pre-incubated for 1 hour with LY29400 0 ⁇ M (0), 5 ⁇ M (5), 25 ⁇ M (25), 50 ⁇ M (50) or 100 ⁇ M (100), as indicated, followed by stimulation for additional 20 minutes (+) by 25 ⁇ g/ml F3 (F3) or no stimulation (-) as indicated, followed by measuring the phosphorylation of JNK; the detected expression of p-JNK, JNK, p-p38 and p38 is indicated by arrows on the right;
  • Fig. 42 shows the results of a Western blot analysis with anti-IL-1 antiserum of whole cells lysates of J774A.1 cells (J774A.1 cells) pre-incubated for 1 hour (+) with 50 ⁇ M LY294002 (LY294002) or not pre-incubated (-) as indicated, followed by stimulation for additional 6 h (+) by 25 ⁇ g/ml F3 (F3) or no stimulation (-) as indicated; the detected expression of pro-IL-1 and actin (as an internal control) is indicated by arrows on the right; Fig.
  • CD14+ shows a diagram illustrating a flow cytometric analysis of CD14 + monocyte/macrophage (CD14+), CD14 + CD26- monocyte/macrophage (CD14+ CD26-) or CD14 + CD26+ monocyte/macrophage (CD14+ CD26+) grown in colture in presence of 100 ⁇ g/mL LPS, (100 ⁇ g/mL LPS), 10 ⁇ g/mL F3 (10 ⁇ g/mL F3) or 100 ⁇ g/mL F3 (100 ⁇ g/mL F3), as indicated; the x axis shows the administered concentration of F3 and LPS; the y axis shows the ratio of the cells expressing the indicated marker compared to the control;
  • Fig. 44 shows a diagram illustrating a flow cytometric analysis of CD83 + dendritic cells (CD83+), CD83 + CD26- dendritic cells (CD83+ CD26-) or CD83 + CD26+ dendritic cells (CD83+ CD26+) grown in colture in presence of 100 ⁇ g/mL LPS, (100 ⁇ g/mL LPS), 10 ⁇ g/mL F3 (10 ⁇ g/mL F3) or 100 ⁇ g/mL F3 (100 ⁇ g/mL F3), as indicated; the x axis shows the administered concentration of F3 and LPS; the y axis shows the ratio of the cells expressing the indicated marker compared to the control; Fig. 45 shows a diagram illustrating a flow cytometric analysis of CD56 +
  • NK cells CD56+
  • CD56 + CD16- NK cells CD56 + CD16-
  • CD56 + C16+ NK cells CD56+ CD16+
  • the x axis shows the administered concentration of F3 and LPS;
  • the y axis shows the ratio of the cells expressing the indicated marker compared to the control;
  • Fig. 46 shows a diagram illustrating a flow cytometric analysis of CD34 + hematopoietic cells (CD34+), CD34 + CD45- hematopoietic cells (CD34 + CD45-) or CD34 + C45+ hematopoietic cells (CD34+ CD45+) grown in colture in presence of 100 ⁇ g/mL LPS, (100 ⁇ g/mL LPS), 10 ⁇ g/mL F3 (10 ⁇ g/mL F3) or 100 ⁇ g/mL F3 (100 ⁇ g/mL F3), as indicated; the x axis shows the administered concentration of F3 and LPS; the y axis shows the ratio of the cells expressing the indicated marker compared to the control;
  • Fig. 47 shows a diagram illustrating the results of an XTT assay performed on human umbilical cord blood MNC proliferation treated with F3 (F3) or LPS (LPS) at 100 ⁇ g/mL (100 ⁇ g/mL), 50 ⁇ g/mL (50 ⁇ g/mL), 25 ⁇ g/mL (25 ⁇ g/mL), 12.5 ⁇ g/mL (12.5 ⁇ g/mL), 6.5 ⁇ g/mL (6.5 ⁇ g/mL), 3.125 ⁇ g/mL (3.125 ⁇ g/mL), 1.56 ⁇ g/mL (1.56 ⁇ g/mL), 0.78 ⁇ g/mL (0.78 ⁇ g/mL) and 0.39 ⁇ g/mL (0.39 ⁇ g/mL), or untreated (Controls) human umbilical cord blood MNC proliferation, as indicated; the x axis shows the concentration of F3 administered; the y axis shows the ratio of the cells expressing the indicated marker compared to
  • Fig. 48 shows a diagram illustrating the effect of F3 on CD56 + NK-cell mediated cytotoxicity upon pre-incubation of NK-cells with F3 for 7 days at at 100 ⁇ g/mL (100 ⁇ g/mL), 50 ⁇ g/mL (50 ⁇ g/mL), 25 ⁇ g/mL (25 ⁇ g/mL), 12.5 ⁇ g/mL (12.5 ⁇ g/mL), 6.5 ⁇ g/mL (6.5 ⁇ g/mL), 3.125 ⁇ g/mL (3.125 ⁇ g/mL) or untreated (Controls) as indicated; the x axis shows that K562 cell lysis was assessed by Alamar Blue assay at an effector/target ratio of 20:1 ; the y axis shows results expressed as a percentage of survival to the control; Fig.
  • 49 shows a diagram illustrating the effect of LPS on CD56 + NK-cell mediated cytotoxicity upon pre-incubation of CD56 + NK-cells with LPS for 7 days at at 100 ⁇ g/mL (100 ⁇ g/mL), 50 ⁇ g/mL (50 ⁇ g/mL), 25 ⁇ g/mL (25 ⁇ g/mL), 12.5 ⁇ g/mL (12.5 ⁇ g/mL), 6.5 ⁇ g/mL (6.5 ⁇ g/mL), 3.125 ⁇ g/mL (3.125 ⁇ g/mL) or untreated (Controls) as indicated; the x axis shows that K562 cell lysis was assessed by Alamar Blue assay at an effector/target ratio of 20:1 ; the y axis shows results expressed as a percentage of survival to the control;
  • Fig. 50 shows a diagram illustrating the effect of F3 on CD56 + NK-cell mediated cytotoxicity upon pre-incubation of NK-cells with with F3 for 7 days at at 100 ⁇ g/mL (100 ⁇ g/mL), 50 ⁇ g/mL (50 ⁇ g/mL), 25 ⁇ g/mL (25 ⁇ g/mL), 12.5 ⁇ g/mL (12.5 ⁇ g/mL), 6.5 ⁇ g/mL (6.5 ⁇ g/mL), 3.125 ⁇ g/mL (3.125 ⁇ g/mL) or untreated (Controls) as indicated; the x axis shows that K562 cell lysis was assessed by Alamar Blue assay at an effector/target ratio of 5:1 ; the y axis shows results expressed as a percentage of survival to the control;
  • FIG. 52 shows a diagram illustrating the difference in the induced NK subtypes CD56 + CD3 - (CD56 + CD3 -), CD56+ CD3 + (CD56+ CD3 +), CD 56 (CD 56), in cells pre-treated with 100 ⁇ g/mL F3 versus equal volume of PBS, as indicated; on the x axis six individual volunteers identified with letters a, b, c, d, e, and f are indicated; on the y axis the ratio of the cells expressing the indicated marker compared to the control is indicated;
  • Fig. 53 shows a first chart illustrating the signaling pathways triggered by the proposed binding event of RPS with TLR
  • Fig. 54 shows a second chart illustrating; the signaling pathways triggered by the proposed binding event of RPS with TLR;
  • Fig. 55 shows a diagram illustrating the results of a RT-QPCR performed with a probe specific for Blimp-1 on purified mouse splenic B cells cultured with 2.5 ⁇ g/ml LPS (LPS), 10 ⁇ g/ml Reishi F3 (F3) or left untreated (no treat), wherein cells harvested at day 0 (day 0) or at day 3 (day 3) as indicated; the x-axis shows the time of treatment and substance administered; the y axis shows the folds of Blimp - 1 nRNA induction normalized to L32 mRNA;
  • Fig. 56 shows the results of a Western Blot performed with a Blimp-1 monoclonal antibody and actin antibody as a control, on purified mouse splenic B cells cultured with 2.5 ⁇ g/ml LPS (LPS), 10 ⁇ g/ml Reishi F3 (Reishi F3) or left untreated (no treat), wherein cells harvested at day 0 (day 0) or at day 3 (day 3) treated were lysed and total cell lysates were used for western blot analysis as indicated; first lane no treatment day 0, second lane LPS treated day 3, third lane Reishi F3 day 3, the detected expression of Blimp-1 and actin (as an internal control) are indicated on the right; Fig.
  • LPS 2.5 ⁇ g/ml LPS
  • Reishi F3 Reishi F3
  • left untreated no treat
  • 57 shows a diagram illustrating the results of a RT-QPCR using primer and probe sets to Blimp-1 and normalized to PPIA's expression level on purified human peripheral B cells from different donors (donor #1 ) (donor #2) and (donor #3) were treated with Reishi F3 for 6 days; the x axis shows the time of treatment expressed in days; the y axis shows the detected Blimp-1 expression expressed as folds of Blimp - 1 nRNA induction normalized to PPIA mRNA;
  • Fig. 58 shows a diagram illustrating the results of a RT-QPCR using primer and probe sets to Blimp-1 and normalized to PPIA's expression level on isolated human dendritic cells treated with 10 ⁇ g/ml Reishi F3 (Reishi F3) and not treated (Control) subjected to RT-QPCR at day 7; the x axis shows the substace administered; the y axis shows the detected Blimp-1 expression expressed as folds of Blimp - 1 nRNA induction normalized to PPIA mRNA;
  • Fig. 59 shows the results of a Western Blot performed with a IL-1 monoclonal antibody a, on murine macrophages cultured with F3 (F3), F3G1 (F3G1 -1 , F3G1 -2, F3G1-3), F3G2 (F3G2-1 and F3G2-2), F3G2H1 (F3G2H1 ), F3G2H2 (F3G2H2), F3G2H1A (F3G2H1A), and F3G2H1 B (F3G2H1 B) or left untreated (Control), as indicated; first lane no treatment, second lane F3, third lane F3G2H1 , fourth lane F3G2H2, fifth lane F3G2H1A, sixth lane F3G2H1 B, seventh lane F3G1-1 , eight lane FRG1-2, ninth lane FRG1 -3, tenth lane F3G2-1 , eleventh lane FRG2-2
  • Fig. 60 shows the results of a Western Blot performed with a IRAK monoclonal antibody a, on murine macrophages cultured with F3 (F3) or LPS (LPS) for 0 minutes (0), 10 minutes (10), 20 minutes (20), 30 minutes (30), 60 minutes (60), or 120 minutes (120) as indicated; first lane o minutes, second lanelO minutes, third lane 20 minutes, fourth lane 30 minuets, fifth lane 60 minutes, sixth lane 120 minutes; the substance administrated (F3 or LPS) is indicated on the left; the detected expression of IRAK is indicated on the right;
  • F3 F3
  • LPS LPS
  • Fig. 61 shows a flow chart illustrating a schematic representation of treatment of RAW 264.7 cells with LPS and F3 described in Example 22
  • Fig. 62 shows a schematic representation of the reactions involved in the in vitro Nitrate Assay described in Example 22;
  • Fig. 63 shows a diagram illustrating the effect of Fraction 3 (GL(PS)-Wu or Wu) on Nitrite Oxide LPS induced production in RAW 264.7 cells, subtract the Fraction 3 background; the x-axis shows the Fraction 3 concentration administered to the culture expressed in ⁇ g/mL; the y-axis shows the Nitrite
  • Fig. 64 shows a diagram illustrating the effect of Fraction 3 (GL(PS)_Wu or Wu) on Nitrite Oxide LPS induced production in RAW 264.7 cells, subtract the Fraction 3 background; the x-axis shows the the Fraction 3 concentration administered to the culture expressed in ⁇ g/mL; the y-axis shows the inhibition, expressed in percentage, of Nitrite Oxide production in RAW 264.7 cells treated with the Fraction 3 with or without LPS; and
  • Fig. 65 shows a diagram illustrating the cytotoxic effect of Fraction 3 (GL(PS)_Wu or Wu) on RAW 264.7 cells; the x-axis shows the Fraction 3 concentration administered to the culture expressed in ⁇ g/mL; the y-axis shows the cell viability expressed in percentage, of RAW 264.7 cells treated with
  • a fucose-containing glycoproteic fraction of an extract of Ganoderma Lucidum Reishi refers to a constituent part of the extract that includes at least one of a polysaccharide and a glycopeptide comprising fucose residues.
  • glycoprotein or “glycopeptide” refers to a protein of any length and dimensions with covalently attached sugar units, either bonded via the OH group of serine or threonine O glycosylated) or through the amide NH2 of asparagine (N glycosylated) or portions thereof.
  • polysaccharide refers to a polymers of any length and dimensions comprising monosaccharide residues linked glycosidically in branched or unbranched chains.
  • extract refers to a concentrated preparation obtained by removing active constituents from a given substance; when the active constituents are included in a solvent, the removal of the active constituents can be performed by evaporating all or nearly all the solvent and adjusting the residual mass or powder to a prescribed standard; extracts are usually prepared in three forms, semiliquid or of syrupy consistency, pilular or solid and as dry powder.
  • Ganoderma Lucidum refers to fungus Ganoderma Lucidum or Reishi, any tissue, part or fraction therefrom and/or any preparation thereof including homogenates, suspensions, filtrates, filtration residues and solutions.
  • preparation refers to a composition processed, manufactured, or compounded starting from a given substance
  • concentration preparation refers to a preparation with an increased ratio of the mass or volume of active constituents to the mass or volume of the non-active constituents or to the mass volume of the entire composition, compared with the same ratio in the given substance.
  • fraction refers to one of the separable constituents of a substance.
  • the fucose-containing glycoprotein fraction of Ganoderma Lucidum constitutes an active constituent of the extract of Ganoderman Lucidum, in view of the experimental procedures exemplified in the Examples sections and in particular in Example 5 showing that treatement with fucosidase affects the properties and activities associated with the extract.
  • a possible model to interpret the relationship between enzymatic treatment and bioactivity of the fucose- containing glycoprotein fraction is illustrated in Fig. 12.
  • the activities and properties of the fucose-containing glycoprotein fraction as well as methods and compositions using or including the fucose-containing glycoprotein fraction shall not be limited in scope and applications by the above model included only for explanatory purposes.
  • the fucose-containing glycoprotein fraction is included in a fraction of Ganoderma Lucidum (herein also denominated F3, Fraction 3, EORP, GL(PS)_Wu, or Wu) showing a light absorbance of about 1 .8 O. D. at 625 nm identified and isolated from a water-soluble extract of Ganoderma Lucidum (crude Reishi extract) by experimental procedures exemplified in Example 1 , 6 and 9 (see in part. Fig. 2).
  • a fraction of Ganoderma Lucidum herein also denominated F3, Fraction 3, EORP, GL(PS)_Wu, or Wu
  • Fraction 3 includes a fucose-containing glycoprotein fraction, which comprises terminal fucose residues.
  • the phrase "terminal fucose residues" identifies fucose residues of a chain of sugars located in a region proximate to a free end of a chain of sugars.
  • the fucose-containing glycoprotein fraction of Fraction 3 also includes fucose residues bound with ⁇ 1 ,2-fucosidic linkages and ⁇ 3,4-fucosidic linkages, as established by experimental procedures exemplified in Example 5.
  • fucose-containing glycoprotein fraction of Fraction 3 can also comprise glucose mannose, N-acetylglucosamine, xylose and rhamnose, as established by experimental procedures exemplified in Example 1 and Example 6 (see in part Table 1 and Table Vl).
  • the fucose-containing glycoprotein fraction of Fraction 3 can also include an amino acidic component, as established by experimental procedures exemplified in Example 1.
  • the amino acidic component of Fraction 3 can be significantly modified without impairing the activities associated with the fucose-containing glycoprotein fraction of Fraction 3, as shown by experiments exemplified in Example 5.
  • Fraction F3 can be obtained by a process comprising: homogenizing a plant tissue of Ganoderma Lucidum and/or providing an homogenized plant tissue from Ganoderma Lucidum; extracting the homogenized plant tissue of
  • Extracting refers to any suitable procedure or protcol to provide an extract starting from a given substance; determination of such protocols can be accomplished by those skilled in the art depending upon a variety of variables, including the substance and the active consitutents to be removed from the substance; exemplary procedures include treatment based on different solubility of the constituents of the substance in different solvents.
  • Extracting the homogenized plant tissue can be performed by any suitable procedure or protocol to provide an extract of Ganoderma Lucium including a fucose- containing glycoprotein or fucose-containing polysaccharide constituents from the homogenized plant tissue; for example a suitable procedure includes treating the homogenized plant tissue with aqueous alkalyne solution, for example 0.1 N NaOH, for a predetermined time to form a crude extract.
  • a suitable procedure includes treating the homogenized plant tissue with aqueous alkalyne solution, for example 0.1 N NaOH, for a predetermined time to form a crude extract.
  • filtering refers to any suitable procedure to separate a constituent of a substance, such as an active constituent, from other consitutents of the substance, such as impurities; determination of such protocols can be accomplished by those skilled in the art depending upon a variety of variables, including the substance, the active consitutents and the inactive constituents of the substance; exemplary filtration procedures include dialysis and gel filtration chromatography. Filtering the extracted homogenized plant tissue can be performed by subjecting the crude extract to filtration, such as gel filtration chromatography e.g. using a Sephacryl S-500 column, and eluting with an aqueous solution to form one or more fractions. In one embodiment the aqueous solution is buffered at about pH 7.0, for example a Tris buffer solution. Specific embodiments of the above-mentioned process to obtain Fraction
  • the fucose-containing glycoprotein fraction is included in fractions of Fraction F3 (herein also collectively named Subfractions), herein identified as F3G1 , F3G2, F3G3, and the F3G2 sub-fractions F3G2H1 and F3G2H2.
  • Subfractions are isolated from Fraction 3 by experimental procedures exemplified in Examples 7 and 8.
  • F3G1 shows a light absorance of about 0.4 O. D. at 480 nm
  • F3G2 shows a light absorbance of about 0.1 O. D. at 480 nm
  • F3G2H1 shows a light absorbance of about 0.10 O. D. at 480 nm
  • F3G2H2 shows a light absorbance of about 0.5 O. D. at 480 nm, as established by experimental procedures exemplified in Example 6 (see also Figs. 14 and 15).
  • the fucose-containing glycoprotein fraction comprised in the Subfractions can also include in addition to the fucose residues other sugars such as glucose and mannose galactose, ⁇ /-acetylglucosamine, and xylose as established by esperimental procedures exemplified in Example 6 (see in particular Table IV).
  • the Subfractions F3G1 , FG2 and F3G3 can be obtained by partitioning Fraction 3.
  • partitioning refers to any suitable procedure or protocol to divide a substance in two or more constituents thereof; determination of such protocols can be accomplished by those skilled in the art depending upon a variety of variables, including the substance, and the consitutents to be partitioned.
  • Partitioning Fraction 3 can be performed by filtering Fraction 3, (for example with an anion exchanger such as Diaion-WA30 anion exchanger or by gel filtration chromatography, e.g. on a TSK HW-75 column), and isolating the anion exchanger such as Diaion-WA30 anion exchanger or by gel filtration chromatography, e.g. on a TSK HW-75 column), and isolating the anion exchanger such as Diaion-WA30 anion exchanger or by gel filtration chromatography, e.g. on a TSK HW-75 column), and isolating the
  • Subfractions F3G1 , F3G2 and F3G3 from the filtered Fraction 3, (for example by elution with an alkaline solution, including for example at least one of NaCI).
  • F3G2 subfractions F3G2H1 ad F3G2H2 can be obtained by subjecting F3G2 to further partitioning.
  • partioning of F3G2 can be performed by filtering Sub-fraction F3G2 e.g. by gel filtration chromatography e.g. on a TSK HW-75 column, and isolating the Subfractions F3G2H1 and F3G2H2 from the filtered Subfraction F3G2 e.g. by eluting the the filtered Subfraction F3G2 with an acqueous solution.
  • Exemplary embodiments of the above-mentioned process to obtain the Subtractions are illustrated in Example 6.
  • a method to mediate biological event associated with activated expression of a cytokine in a mammalian cell is disclosed.
  • biological event refers to an occurrence being of or relating to biology or to life and living processes.
  • immediate used intercheangeably with
  • “regulate” and “modulate” refers to specifically controlling, or influencing an item identified thereafter, the item including a molecule, pathway, event or function, wherein “mediate” can include regulation by activation, stimulation, inhibition, alteration or modification of such molecule, pathway event or function.
  • cytokine refers to a process by which the gene's coded information is converted into the cytokine.
  • activate refers to initiating and/or enhancing of a process such as expression of a cytokine, or to converting a compound, such as protein nucleic acid lipid, ion or other compound, from an inactive into an active form or into a different compound, the active form or different compound having a particular biological action.
  • cytokinines refers to proteins or biological factors that are released by cells, such as normal macrophages, fibroblasts, keratinocytes and a variety of transformed cell lines, have specific effects on cell-cell interaction, communication and behaviour of other cells, participate in regulating immunological and inflammatory processes, and can contribute to repair processes and to the regulation of normal cell growth and differentiation; cytokine include interleukins, lymphokines and several related signalling molecules such as TNF and interferons.
  • mammalian cell indicates a cell of mammalian origin, in particular a human or murine, which can be located inside or outside the mammal.
  • the mammal cells can be a human or a murine cell, such as T lymphocytes, human and murine macrophage and murine spleen cells.
  • the cytokine can be IL-1 , IL6, IFN- ⁇ , TNF- ⁇ , IL-12, GM-CSF, G-CSF and/or M-CSF, and the mammalian cell can be a human or a mouse cell, such as T lymphocytes, human and murine macrophage and murine spleen cells.
  • the method to mediate a biological event associated with activated expression of a cytokine in a mammalian cell comprises administering to the mammalian cell an effective amount of the fucose-containing glycoprotein fraction.
  • an effective amount of a compound is at least the minimum amount of the compound that is necessary to minimally achieve, and more preferably, optimally achieve, the desired effect.
  • An effective amount of fucose- containing glycoprotein fraction for use in a given method can be readily determined by one skilled in the art without undue experimentation, depending upon the particular circumstances encountered (e.g. concentrations, cell type and number, etc.) upon reading of the present disclosure and in particular the Examples section.
  • administering refers to any process or protocol suitable to put a coumpound, and in particular the fucose-containing glycoprotein fraction, in contact with the cell, wherein the term “contact” or the phrase “put in contact” mean to place the compound and in particular the fucose-containing glycoprotein fraction and the cell, in a mutual spatial relationship such that a biological interaction between the compound and the cell is feasible; the phrase “biological interaction” refers to the process by which a coumpound and in particular the fucose-containing glycoprotein fraction controls, influences or otherwise affects the normal functioning and/or survival of the cell; determination of such protocols can be accomplished by those skilled in the art depending upon a variety of variables, including the type of cell, whether the contact occurs in vitro, in vivo or ex vivo.
  • Acceptable protocols to administer the fucose-containing glycoprotein fraction include individual dose size, number of doses, frequency of dose administration, and mode of administration, such as topical administration, local administration, or oral administration in vivo, incubation and assays in vitro, or ex vivo administration e.g. to isolated hematopoietic cells, which can be identified by a person skilled in the art upon reading of the present disclosure and, in particular, the Examples section.
  • the effective amount of fucose-containing glycoprotein fraction to mediate a biological event and suitable modes of administration identifiable by a person skilled in the art in view of the biological event to be mediated and the cell where the cytokine expression is activated upon reading of the present disclosure, and in particular the Examples section.
  • the biological event is hematopoiesis
  • the mammalian cell is a hematopoietic cell and the cytokines are GM-CSF, G-CSF, and M-CSF.
  • hematopoiesis refers to the formation of blood cells in a living body, (especially in the bone marrow of mammals).
  • hematopoietic cells refers to a cell involved in the hematopoietic process, such as a B cell, a macrophage, a dendritic cell, and a natural killer cell.
  • the biological event is hematopoiesis
  • the fucose- containing glycoprotein fraction is included in F3, F3G2, F3G2H1 and/or F3G2H1 , possibly in combination with F3G1 and the effective amount to mediate hematopoiesis and suitable modes of administration can be identified by a person skilled in the art upon reading of the present disclosure, in particular the Examples section and Examples 3, 7, 8 and 12.
  • the effective amount to mediate hematopoiesis and suitable modes of administration are identifiable by a person skilled in the art upon reading of the present disclosure on the basis of experiments exemplified in Example 3, 7 and 12.
  • the effective amount to mediate hematopoiesis and suitable modes of administration are identifiable by a person skilled in the art upon reading of the present disclosure on the basis of experiments exemplified in Examples 7 and 8.
  • the biological event is an immunomodulating event
  • the cytokines are IL-1 , IL6, IFN and/or TNF
  • the mammalian cell can be a human or a murine cell, such as T lymphocytes, human and murine macrophage and murine spleen cells.
  • immunomodulating event refers to an occurrence associated with regulation of one or more immune functions, wherein the regulation includes activation, stimulation, inhibition, or alteration or modification of the immune functions; exemplary immunomodulating events are secretion of cytokines, such as lnterleukin-1 IL6 IFN and/or TNF, and/or the secretion precursor thereof, inflammatory response and antitumor activity, in particular the antitumor activity associated with IL-1 IL6, IFN and/or TNF gene expression in a mammalian cell.
  • cytokines such as lnterleukin-1 IL6 IFN and/or TNF
  • the secretion precursor thereof inflammatory response and antitumor activity, in particular the antitumor activity associated with IL-1 IL6, IFN and/or TNF gene expression in a mammalian cell.
  • the effective amount of fucose-containing glycoprotein fraction to be administered to mediate the immunomodulating event associated with the activated expression of a cytokine and suitable modes of administration can be identified by a person skilled in the art in view of the immunomodulating event to be activated, upon reading of the present disclosure and in particular of the procedures disclosed in the Examples, in particular Examples 3, 6, 7, 8 and 12.
  • the immunomodulating event is an inflammatory response
  • the cytokines are IL-1 , IL-6 and TNF- ⁇
  • the fucose-containing glycoprotein fraction can be included in F3, F3G2, F3G2H1 and/or F3G2H2 alone or in combination with F3G1 , and F3G3.
  • the phrase "inflammatory response" refers to a series of biological events elicited by the immune system of an organism upon lesion, injury or destruction of part of the organism, the series of events serving to destroy, dilute or wall off (sequester) both the injurious agent and the injured part; in organism such as mammals the biological events include biochemical and histological events, such as production of proteins dilatation of arterioles, capillaries and venules, with increased permeability and blood flow, exudation of fluids, including plasma proteins and leucocytic migration into the inflammatory focus.
  • the effective amounts to mediate the inflammatory response and suitable modes of administration can be identified upon reading of the present disclosure on the basis of experiments exemplified in Examples 3, 7 and 12.
  • the fucose-containing glycoprotein fraction is included in one or more of the Subfractions and the effective amounts to mediate the inflammatory response related procedures of administration can be identified on the basis of experiments exemplified in Example 7 and 8.
  • the immunomodulating event is antitumor activity
  • the cytokines are INF- ⁇ and TNF- ⁇
  • the fucose-containing glycoprotein fraction is comprised in F3, F3G2, F3G2H1 and/or F3G2H2 alone or in combination with
  • F3G1 , and F3G3 and the mammalian cell is a human or a mouse cell, such as T lymphocytes, human and murine macrophage and murine spleen cells.
  • antiitumor activity refers to a series of biological events elicited by the immune system to counteract tumor formation or growth.
  • the effective amounts to mediate the antitumor activity and related procedures of administration can be identified on the basis of experiments exemplified in Examples 3, 7 and 12.
  • the fucose-containing glycoprotein fraction is comprised in one or more of the Subfractions the effective amounts to mediate the antitumor activity and related procedures of administration can be identified upon reading of the present disclosure on the basis of experiments exemplified in Example 7 and 8.
  • the fucose containing glycoprotein fraction can be used in a method to stimulate the expression of an inflammatory cytokine in a mammalian cell, such as human or mouse cell.
  • Inflammatory cytokines comprise, IL-1 IL6 and TNF.
  • the phrase "inflammatory cytokine” refers to a cytokine whose expression is associated with elicitation of an inflammatory response in an organism.
  • the method comprises administering to the cell an effective amount of the fucose-containing glycoprotein fraction, in particular comprised in each of the Fractions alone or combination with each other.
  • a method to induce a Toll-Like Receptor (TLR) mediated event in a mammalian cell, such as a human or mouse cell is disclosed.
  • a TLR mediated event is a biological event mediated by one or more of the receptors belonging to the Toll-Like Receptors family (TLRs family).
  • the TLR mediated event can be an event mediated by TLR4 a member of the TLRs family (Muzio and Mantovani, 2000; Akira et al 2001 ; Hsu and Wen 2002; Mambula et al 2002; Aderem and Ulevitch 2000; Brown et al. 2002).
  • the TLR mediated event can be the production of an inflammatory cytokine in particular the inflammatory cytokine lnterleukin-1 , including up- regulation of IL-1 secretion and pro-IL-1 (precursor of IL-1 ) as well as IL-1- converting enzyme expression.
  • the TLR-mediated event also include phosphorylation and/or degradation of proteins involved in the TLR4 signaling pathways such as the IL-1 R associated kinase (IRAK), a serine/threonine kinase, also recruited to the TLR signaling complex.
  • IRAK IL-1 R associated kinase
  • serine/threonine kinase also recruited to the TLR signaling complex.
  • the method to induce a TLR mediated event comprises administering to the cell an effective amount of the fucose-containing glycoprotein fraction, in particular comprised in F3 and/or at least one of the Subfractions F3G1 , F3G2, F3G2H1 , F3G2H2, F3G2H1A, and F3G2H1 B
  • the effective amount to induce a TLR-mediated event and suitable modes of administration can be identified by a person skilled in the art upon reading of the present disclosure, in particular the Examples section and Examples 10, 11 and 12 (see also Example 7 and Figs. 20-21 , 23-25 and 59-60).
  • a method to modulate a protein kinase pathway in a mammal cell the protein kinase pathway associated with inflammatory cytokine lnterleukin 1 , such as a human or murine cell, is disclosed.
  • a “protein kinase pathway” refers to a signal transduction pathway including the activation of at least one protein kinase;
  • a signal transduction pathway refers to a series of biochemical reactions including at least one reaction, directed to transmit from one portion of a cell to another, a signal generated by the interaction of the cell with a compound, the signal resulting in a cellular response.
  • the protein kinase pathway can be a pathway including the activation of the protein kinases PKC, MEK1 , PAK, Rac-1 and/or MAPKs, such as ERK, JNK, and p38 (PKC: protein kinase C, MEK1 : mitogen-activated protein kinase/extracellular signal-regulated kinase, PAK: p21 -activated kinase; ERK: extracellular signal-regulated kinase; JNK: c-Jun N-terminal kinase; p38: p38- mitogen activated protein kinase), and the associated transduction of diverse signals resulting in different biochemical reactions and cellular responses, such as multiple cytokine productions or production of specific cytokine such as IL-1.
  • PKC protein kinase C
  • MEK1 mitogen-activated protein kinase/extracellular signal-regulated kinase
  • PAK p21
  • the protein kinase pathway can include a variety of compounds, including molecules, involved in the transmission of the signal from one portion of the cell to another, exemplary molecules involved are: cell surface receptors, i.e. molecules and complexes of molecules involved in the transmission of the signal across the plasma membrane of a cell, and intracellular signal transduction molecules, i.e. molecules or complexes of molecules involved in the transmission of the signal from the plasma membrane of a cell through the cytoplasm of the cell, and/or into the cell's nucleus. Other compounds such as proteins, lipids nucleic acid or ions can be involved.
  • the compounds are activated in the pathway to transduce the signal wherein the active form or different compound resulting from the activation has the function of transducing the signal and/or to convert the signal into a cellular response.
  • the method comprises administering to the mammalian cell an effective amount of a fucose-containing glycoprotein fraction, in particular comprised in
  • F3 the effective amount to modulate the protein kinase pathway and suitable modes of administration identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Examples section and of Examples 1 1 to 15.
  • the effective amount of the fucose-containing glycoprotein fraction can be an amount activating PKC, MEK1 , PAK and/or MAPKs in the cell, the activated PKC, MEK1 , PAK and/or MAPKs leading to multiple cytokine production.
  • the effective amount of the fucose-containing glycoprotein fraction can be an amount activating a PKC/MEK1/ERK signal transduction pathway the activated pathway resulting in a transcriptional, posttranscriptional and posttranslational regulation of the lnterleukin 1 gene expression.
  • the fucose-containing glycoprotein fraction comprised in at least one of the Subfractions modulates one or more of the above protein kinase pathways associated with inflammatory cytokine lnterleukin 1 , can be envisioned by a person skilled in the art based on the ability of the Subfractions to induce IL-1 expression upon reading of the present disclosure, in particular Examples 11 to 15 in view of Example 7.
  • an effective amount of fucose-containing glycoprotein fraction is administered to mediate a pathway PTK/PKC/MEK1/ERK, i.e. a pathway that includes PTK and ERK, compounds which are activated upstream PTK, compounds which are activated between PTK and ERK, including PKC and MEK with PKC activated upstream MEK, and compounds which are activated downstream of ERK.
  • a pathway PTK/PKC/MEK1/ERK includes PTK-mediated phosphorylation, followed by induction of PKs and activation of MAPKs: ERK, JNK, and p38.
  • the pathway includes PTK ⁇ PKC ⁇ MEK1 ⁇ ERK, wherein one arrow indicates that no compound is activated between the compound distal to the arrowhead and the compound proximate to the arrowhead, and two arrows indicate that one or more compounds are activated in the pathway between the compound distal to arrowhead and the compound proximate to the arrowhead.
  • an effective amount of F3 is administered to mediate a pathway PTK/Rac1/PAK/p38, i.e. a pathway that includes PTK and p38, compounds which are activated upstream PTK, compounds which are activated between PTK and p38, including Rad and PAK, with Rac1 activated upstream PAK, and compounds which are activated downstream of p38.
  • the pathway PTK/Rac1/PAK/p38 includes PTK-mediated phosphorylation, followed by induction of Rad , PAK and activation of MAPKs: JNK, and p38, with Rad and PAK activated upstream of JNK and p38.
  • the pathway comprises the pathway PTK ⁇ Rac1 ⁇ PAK ⁇ p38.
  • the pathway comprises PTK ⁇ Rad ⁇ PAK ⁇ JNK.
  • an effective amount of F3 is administered to mediate a PI3K signaling pathway, the effective amount and suitable modes of administration identifiable by a person skilled in the art upon reading of the present disclosure in particular the Examples section, and Example 15.
  • PI3K -signaling pathway refers to a signal transduction pathway in which PI3K protein regulates a signal transduction pathway that is substantially independent of a pathway in which PI3K protein regulation converges with common members of a pathway involving for example PTK and ERK. More particularly, a PI3K-signaling pathway includes components downstream of PI3K proteins and continues downstream in a series of signal transduction events.
  • Figs. 54 and 55 Schematic representations of the protein kinase pathways are illustrated in Figs. 54 and 55.
  • the activities and properties of the fucose-containing glycoprotein fraction as well as methods and compositions using or including one or more the fucose-containing glycoprotein fraction shall not be limited in scope and applications by the above model included only for explanatory purposes.
  • a method to modulate the differentiation of a mononuclear cell comprising administering an effective amount of the fucose-containing glycoprotein fraction to the cell, the effective amount identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Examples section and Example 16.
  • differentiate refers to the process cells undergo as the cells mature into a distinct cell type, wherein differentiated cells have distinctive characteristics, perform specific functions and are less likely to divide.
  • cell type refers to a distinct morphological or functional form of a cell.
  • the differentiation of a cell can be characterized by phenotypic changes, wherein the "phenotype” of a cell refers to the total characteristics displayed by the cell, under a particular set of environmental factors, the characteristics resulting from interaction between the genotype and the environment, and a "mature phenotype” is the phenotype displayed by the cell complete in natural growth or development.
  • phenotypic changes refers to modifications of the phenotype of the cell; the phrase “immuno-phenotipic changes” refers to modification of the the phenotype of the cell affecting immune functions associated with the cell.
  • an effective amount of the fucose-containing glycoprotein fraction comprised in Fraction 3 is administered to the mononuclear cell to induce immuno-phenotypic changes of the mononuclear cell, the immuno- phenotypic changes characterize the differentiation of the mononuclear cell in monocyte/macrophage, such as CD14 + CD26 + expression, and the effective amount is identifiable by a person skilled in the art upon reading of the present disclosure, on the basis of experiments exemplified in Example 16.
  • an effective amount of the fucose-containing glycoprotein fraction comprised in Fraction 3 is administered to the mononuclear cell to induce immuno-phenotypic changes of the mononuclear cell, the immuno- phenotypic changes characterize the differentiation of the mononuclear cell in dendritic cells, such as CD83 + CD1a + expression, and the effective amount is identifiable by a person skilled in the art upon reading of the present disclosure, on the basis of experiments exemplified in Example 16.
  • an effective amount of the fucose-containing glycoprotein fraction comprised in Fraction 3 is administered to the mononuclear cell to induce immuno-phenotypic changes of the mononuclear cell, the immuno- phenotypic changes characterize the differentiation of the mononuclear cell in NK cells, such as CD16 + CD56 + expression, and the effective amount is identifiable by a person skilled in the art upon reading of the present disclosure, on the basis of experiments exemplified in Example 16.
  • a method to activate the expression of MHCII, CD80 and/or CD86 in primary monocytes and/or dendritic cells comprising administering an effective amount of the fucose-containing glycoprotein fraction to the monocytes and/or dendritic cells, the effective amount identifiable by a person skilled in the art upon reading of the present disclosure and in particular of the Examples section and Example 19.
  • cytotoxicity refers to the quality of being toxic to cells proper of NK cells, including, for example, antibody-dependent cell-mediated reactions, or antibody-independent cell- mediated reactions .
  • the method comprising administering to the NK cell an effective amount of the fucose-containing glycoprotein fraction, for example comprised in at least one of the Fractions, the effective amount identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Example section and Examples 17 and 18.
  • the NK cell can be a CD3 + CD56 + NKT cell or CD3OD56 + NK cell.
  • the effective amount of the fucose-containing glycoprotein fraction not only enhances the cytotoxicity of an NK cell, e.g. CD56+ NK-cells, against an NK-sensitive tumor cell, e.g. tumor-cell line K562, but also mediate the expression of cell surface markers.
  • An effective amount of fucose- containing glycoprotein fraction and suitable modes of administration to achieve those effects, is identifiable by a person skilled in the art upon reading of the present disclosure on the basis of experiments exemplified in Examples 17 and 18.
  • the effective amount of the fucose-containing glycoprotein fraction also results in a modified proportion between NKT cells and UCB cells, wherein the proportion of NKT cells of UCB decreases, while the proportion of NK-cells increases, after treatment with the fucose-containing glycoprotein fraction.
  • An effective amount of fucose-containing glycoprotein fraction and suitable modes of administration to achieve those effects, is identifiable by a person skilled in the art upon reading of the present disclosure on the basis of experiments exemplified in Example 18.
  • a method to activate the expression of one or more cytokines in a mammalian cell, such as human or mouse cells is disclosed.
  • the mammalian cells can be a human or a mouse cell, such as T lymphocytes, human and murine macrophage and murine spleen cells.
  • the one or more cytokines include IL-1 , IL6, IFN- ⁇ , TNF- ⁇ , IL-12, GM- CSF, G-CSF and M-CSF and the method comprises administering to the mammal cell an effective amount of the fucose-containing glycoprotein fraction.
  • an effective amount of the fucose-containing glycoprotein fraction for activating the expression of one or more cytokines and suitable modes of administration are identifiable by a person skilled in the art in view of the cytokine/cytokines whose expression is to be activated and the cell/cells wherein the expression is to be activated, upon reading of the present disclosure, in particular the Examples section and Examples 3, 7, 8 and 12.
  • the cytokines are IL-1 , IL6, IFN, and TNF- ⁇
  • the fucose-containing glycoprotein fraction is comprised in F3, F3G1 , F3G2 F3G3, F3G2, F3G2H1 and/or F3G2H2, and the effective amount and suitable modes of administration are identifiable by a person skilled in the art upon reading of the present disclosure in particular the Examples section and Examples 3, 7, 8 and 12.
  • the cytokines are IL-1 , IL6, IFN- ⁇ , and TNF- ⁇ , IL- 12, GM-CSF, G-CSF and/or M-CSF, the fucose-containing glycoprotein fraction is comprised in F3, and the effective amount and suitable modes of administration are identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Examples section and Examples 3, 7 and 12.
  • the cytokines are IL-1 , IL-6, IFN, TNF- ⁇ , I L-12 and G-CSF, the fuose containing glycoprotein fraction is comprised in F3G1 , F3G2,
  • F3G2H1 and/or F3G2H1 are identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Examples section and Examples 7 and 8.
  • the cytokines are IL-12 GM-CSF, G-CSF and M- CSF
  • the fucose containing glycoprotein fraction is comprised in F3G2, F3G2H1 and/or F3G2H1 , and the effective amount and suitable modes of administration are identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Examples section and Example 7 and 8.
  • the cytokines are IL-1 , IL-12, TNF- ⁇ and G-CSF
  • the fucose containing glycoprotein fraction is comprised in F3G1
  • the effective amount and suitable modes of administration are identifiable by a person skilled in the art upon reading of the present disclosure, in particular the
  • the cytokines are IL-1 , IL6, IL-12, IFN- ⁇ , TNF- ⁇ , GM-CSF, G-CSF and M-CSF, the fucose containing glycoprotein fraction is comprised in F3G2, and the effective amount and suitable modes of administration are identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Examples section and Example 7 and 8.
  • the cytokines are IL-1 and TNF- ⁇ , the fucose containing glycoprotein ' fraction is comprised in F3G3, and the effective amount and suitable modes of administration are identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Examples section and Example 7 and 8.
  • a method to activate the expression of Blimp-1 a master regulator for plasma cell differentiation (Calame, et al. 2003) in a mammal cell such as a mouse mature B cell and human mature dendritic cell, is disclosed.
  • the method comprises administering an effective amount of a fucose-containing glycoprotein fraction, in particular comprised in F3 and/or F3G2, wherein the effective amount is identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Example sections and Examples 20 and 21.
  • an effective amount of glycoprotein fraction comprised in F3 is able to activate the expression of Blimp-1 , in mature mouse splenocytes, the effective amount and suitable modes of admininstration identifiable by a person skilled in the art upon reading of the present disclosure and in particular of Example 20.
  • a method to inhibit lipopolysaccharide (LPS) induced nitric oxide production in macrophages comprises administering to the macrophage an effective amount of a fucose- containing glycoprotein fraction, in particular comprised in Fraction 3, wherein the effective amount is identifiable by a person skilled in the art upon reading of the present disclosure, in particular the Example sections and Example 22.
  • a method to activate spleen cells proliferation is disclosed, the method comprising administering to the cells an effective amount of the fucose-containing glycoprotein fraction, and in particular F3, wherein the effective amounts and route of administration can be identified by a person skilled upon reading of the present disclosure, in particular the Examples section and Example 2.
  • a method to modify the proteome of a spleen cell comprising administering to the cells an effective amount of the fucose-containing glycoprotein fraction, in particular comprised in F3, wherein the effective amounts and route of administration can be identified by a person skilled upon reading of the present disclosure, in particular the Example section and Example 4.
  • the fucose-containing glycoprotein fraction can be included in a composition together with additional active agents, carriers, vehicles or auxiliary agents identifiable by a person skilled in the art upon reading of the present disclosure.
  • active agent refers to any compound able of a biological interaction with a mammalian cell.
  • vehicle refers to a more or less inert substance added to a prescription in order to confer a suitable consistency or form to the composition.
  • carrier refers to a substance able to transport the fucose-containing glycoprotein fraction in at least one process or protocols suitable for administering the fucose- containing glycoprotein fraction to a mammalian cell.
  • auxiliary agent refers to a compound suitable to assist, enhance or otherwise adjuvate the fucose containing glycoprotein fraction in at least one of the methods herein disclosed.
  • various compositions can be envisioned, each including the fucose-containing glycoprotein fraction in one of the above mentioned effective amounts.
  • kits of parts can be envisioned by the person skilled in the art, the kits of parts to perform at least one of the methods herein disclosed, the kit of parts comprising two or more compositions, the compositions comprising alone or in combination an effective amount of fucose-containing glycoprotein fraction according to the at least one of the above mentioned methods.
  • kits possibly include also compositions comprising active agents other than the fucose-containing glycoprotein fraction, identifiers of a biological event, or other compounds identifiable by a person skilled upon reading of the present disclosure and in particular the Examples section.
  • identifier refers to a molecule, metabolite or other compound, such as antibodies, DNA or RNA oligonucleotides, able to discover or determine the existence, presence, or fact of or otherwise detect a biological event under procedures identifiable by a person skilled in the art;
  • exemplary identifiers are antibodies, Greiss reagents and oligonucleotides as described in the examples, exemplary procedures are western blot, nitrite assay and RT-PCR, as described in the Examples.
  • Exemplary biological events are cytokine expression or other immunomodulating events; an exemplary active agent other than the fucose-containing glycoprotein fraction is LPS.
  • the kit can also comprise at at least one composition comprising an effective amount of fucose-containing glycoprotein fraction and/or a cell line.
  • the compositions and the cell line of the kits of parts to be used to perform the at least one method herein disclosed according to procedure identifiable by a person skilled in the art.
  • Crude Reishi extract (prepared via alkaline extraction (0.1 N NaOH), neutralization and ethanol precipitation) was obtained from Pharmanex Co. (CA, USA). Twenty-eight mg of the crude extract were dissolved in 2 ml_ of Tris buffer (pH 7.0, 0.1 N) and centrifuged to remove the insoluble materials (7 mg). The supernatant was purified by gel filtration chromatography using a Sephacryl S- 500 column (100x1.6 cm) with 0.1 N Tris buffer (pH 7.0) as the eluent. The flow rate was set at 0.5 mL/min, and the elute (7.5 ml_ per tube) was collected. Five fractions were collected (fractions 1-5), each dialyzed to remove excessive salt and lyophilized to give 1.0 mg, 6.2 mg, 5.3 mg, 2.1 mg, and less than 1 mg, respectively.
  • Fractions 1 to Fractions 5 are identifiable as it follows: Fraction 1 : 100-130 mL; Fraction 2: 130-155 ml_; Fraction 3: 155-205 mL; Fraction 4: 205-220 mL; Fraction 5: 220-255 mL.
  • Fraction 3 The main fraction having a light absorbance of about 1.8 at O. D. 625 was designated as Fraction 3 (Fig. 2).
  • the crude extract and each of the isolated fraction were subjected -to anthrone analysis (Somani et al 1987; Jarmyn 1975; Halhoul and I. Kleinberg 1972) to detect sugar components.
  • the polysaccharide extracts/fractions were methanolyzed with 0.5 M methanolic-HCI (Supelco) at 80 0 C for 16 h, re-N- acetylated with 500 ⁇ L of methanol, 10 ⁇ L of pyridine and 50 ⁇ l_ of acetic anhydride, and then treated with the Sylon HTP® trimethylsilylating reagent (Supelco) for 20 min at room temperature, dried and redissolved in hexane.
  • methanolic-HCI Suddenoxane
  • GC- MS analysis of the trimethylsilylated derivatives was carried out using a Hewlett- Packard (HP) Gas Chromatograph 6890 connected to a HP 5973 Mass Selective Detector. Samples were dissolved in hexane prior to splitless injection into a HP-5MS fused silica capillary column (30 m*0.25 mm I. D., HP). The column head pressure was maintained at around 8.2 psi to give a constant flow rate of 1 mL/min using helium as carrier gas. Initial oven temperature was held at 60 0 C for 1 min, increased to 140 0 C at 25 C/min, to 250 0 C at 5 °C/min, and then increased to 300 0 C at 10 °C/min.
  • HP Hewlett- Packard
  • High-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) analysis confirmed that F3 includes a glycoprotein or polysaccharide comprising fucose residues.
  • Example 2 Mitoqen-induced proliferation and colorimetric MTT assay of spleen cells treated with Reishi extract F3
  • Mouse spleen cells treated with Reishi extracts were tested by Mitogen- induced proliferation and colorimetric MTT assay (Mosmanni 1983).
  • whole spleen cells were harvested from BALB/c male mice (6 weeks old), suspended in RPMI-1640 medium containing 10% FCS (fetal calf serum), and centrifuged to remove the supernatant.
  • FCS fetal calf serum
  • the collected precipitated cells were first suspended in 1 mL of RBC lysis buffer (8% NH4CI), then 14 ml_ more of the same lysis buffer were added to destroy red blood cells.
  • the solution was diluted with 15 mL RPMI-1640 medium to stop the reaction, centrifuged to collect the cells, and adjusted the cell final concentration to 2x106 cells/mL with RPMI-1640 medium.
  • Concanavalin A Con A, final concn: 1 ⁇ g/mL
  • the cells were incubated with or without a Reishi extract (or partially purified fraction) in 96-well ELISA plates at 37 0 C with 5% CO2 for 72 h.
  • the cell proliferation was measured based on the MTT assay.
  • MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was dissolved in phosphate buffered saline (PBS) at 5 mg/mL and filtered to sterilize and remove a small amount of insoluble residue present in some batches of MTT. At the times indicated below, the MTT solution (25 ⁇ l_) was added to each well, and plates were incubated at 37 0 C for 4 h. Acid-isopropanol (100 ⁇ l_ of 0.04 N HCI in isopropanol) was added to all wells and mixed thoroughly to dissolve the dark blue crystals.
  • PBS phosphate buffered saline
  • Figs. 3 and 4 show that cell proliferation activity of mouse splenocytes was significantly enhanced by treatment with Fraction 3 and slightly enhanced by treatment with Fraction 2, compared with the control experiment (untreated samples).
  • the optimal concentration of Fraction 3 was between 0.01 and 0.1 ⁇ g/mL and the optimal concentration of crude Reshi extract was between 0.1 and 1.0 ⁇ g/mL, (Figs. 3 and 4).
  • mice spleen cells were aseptically removed from healthy mice (BALB/c male mice, 6 weeks old), adjusted to an ideal cell concentration (4*106 cells/mL) and incubated in RPMI-1640 medium containing 10% of FCS (fetal calf serum) at 37 °C with 5% CO2. After 6 h, the cells were subjected to RNA extraction using Qiagen RNAeasy mini kit to obtain 1 ⁇ g of the desired RNA. Reverse transcription (RT) was performed using the Thermoscript R/T PCR System, and the Thermoscript system protocol I, from Gibco BRL.
  • RT Reverse transcription
  • the reaction was carried out as follows: 8 ⁇ l_ of RNA, 2 ⁇ l_ of primer [Oligo(dT)20], 2 ⁇ l_ of 10 mM dNTP Mix, and DEPC H2O (0.1% diethylpyrocarbonate-treated H2O) was added to each tube, which was then incubated at 65 0 C for 5 min and immediately put on ice.
  • Each sample (3 ⁇ L) was added to each reaction tube and the following reagents were added as a 47 ⁇ L mix: 5 ⁇ L of 1OxPCR buffer, 4 ⁇ L of 10 mM dNTP Mix, 2 ⁇ L of each primer (10 OD/mL, sense and anti-sense), 33 ⁇ L of DEPC H2O, and 1 ⁇ L of ProZyme® (DNA polymerase, from PROtech Technology).
  • the reaction tubes were placed in a Strategene PCR Robocycler (Gradient 96) and run under the following condition: 1 cycle at 92 0 C for 2 min (initial denaturation), then 30 consecutive cycles of 91 0 C for 10 s (denaturation), 59 0 C for 25 s (primer annealing) and 72 0 C for 25 s (primer extension).
  • the reactions were analyzed by gel electrophoresis.
  • cytokines tested i.e. 1L-1 , IL-2, lFN- ⁇ , TNF- ⁇ , IL-4 and IL-6—the first three were significantly expressed by mouse spleen cells treated with Fraction 3 (10 ⁇ g/mL), the expression of the cytokines compared with the expression of a house keeping gene (hypoxanthine phosphoribosyltransferase).
  • a house keeping gene hypoxanthine phosphoribosyltransferase
  • none of the cytokines was detected when cells were treated with the same concentration (10 ⁇ g/mL) of crude Reishi extract (RCE).
  • RCE crude Reishi extract
  • Reishi extract-treated mouse spleen cells were lysed in 350 ⁇ l_ of lysis buffer containing 8 M Urea, 2% CHAPS, 65 mM DTE, 2% v/v isocratic pH gradient (IPG) buffer pH 3-10 NL (non-linear), and a trace of bromophenol blue.
  • the sample was centrifuged for 10 min at 13,000 rpm. The total protein concentration in the sample was measured using Bio-Rad protein concentration assay kit. Samples equal to 500 ⁇ g of proteins were loaded on immobilized pH gradient strips (pH 3-10 NL, 18 cm) for 2-dimensional electrophoresis.
  • the isoelectric focusing was carried out in an IPGPhor apparatus (Amersham Pharmacia Biotech).
  • the second dimension was done in 10-15% polyacrylamide gradient gels using the Protean Il *L 2D multi cell (Bio-Rad). Protein spots were stained with fluorescence dye Sypro RubyTM (Molecular Probes).
  • Sypro Ruby-stained gels were scanned with fluorescence laser scanner (Bio-Rad) generating 10 Mb image. The images were analyzed with ImageMasterTM software (Amersham Pharmacia Biotech). For each gel, the spots were detected and quantified automatically, using default spot detection parameters. Manual spot editing was performed in agreement with the visual inspection of the gels. The relative volume was calculated in order to correct any differences in protein loading and gel staining.
  • the Sypro Ruby stained gels were also subjected to MALDI-TOF MS analysis.
  • the mixture was allowed to dry at ambient temperature. Positive-ion mass spectrum was measured on a MALDI reflection time-of-flight mass spectrometer MALDI (Micromass UK, Manchester, UK) equipped with a nitrogen laser. The reported spectra were accumulated from 50 to 100 laser shots.
  • Figs. 8 to 11 show that treatment of mouse spleen cells with Fraction 3 (10 ⁇ g/mL) resulted in a significant change of the proteome.
  • Fig. 8 (no treatment, control) and Fig. 9 (treatment with Fraction 3) show 623 and 568 detectable spots in the 2-D electrophoresis, respectively.
  • Example 5 Activity of Reishi extracts treated with ⁇ 1 ,2- or ⁇ 1 ,3/4- fucosidases
  • Fraction 3 was treated with protease K to partially destroy the protein component. The results show that proliferation of Con A-stimulated spleen cells remained the same.
  • the active component is a polysaccharaide or glycopeptide containing terminal fucose residues with ⁇ 1 ,2- linkages.
  • the main active component is a glycoprotein containing essential termial fucose residues with ⁇ 1 ,2-linkages.
  • a possible and non-binding model is shown in the chart illustrated in Fig. 12.
  • Example 6 Preparation and purification of Reishi extracts F3 - F3G1 FRG2, F3GH1 , and F3GH2 Crude Reishi extract (prepared via alkaline extraction (0.1 N NaOH), neutralization and ethanol precipitation) was obtained from Pharmanex Co., (CA, USA). All the chemicals and reagents were from Sigma Co., (St. Louis, MO, USA) unless indicated.
  • each fraction was subjected to anthrone analysis or the phenol-sulfuric acid method as described in Example 1 above, to detect sugar components.
  • Five fractions were collected (fractions 1-5), each dialyzed to remove excessive salt and lyophilized, to give 450 mg of each fraction and in particular of F3.
  • F3 was further subjected to a column of Diaion-WA30 anion exchanger (CI " form, 40 * 3.5 cm) eluted with 0.2 and 0.8 M NaCI at a flow rate of 0.5 mL/min, and two fractions were designated as F3G1 (1 1 % yield based on F3) and F3G2 (10% yield based on F3), respectively, as shown in Fig. 14. Another fraction (F3G3, 1 1 % yield based on F3) was generated when the column was further eluted with 2 M NaOH.
  • Diaion-WA30 anion exchanger CI " form, 40 * 3.5 cm
  • the carbohydrate composition of the F3G1 , F3G2 and F3G3 was determined by anthrone colorimetric method and TMS method. The results are shown in Table IV below.
  • Fraction 3 and the subfractions F3G1 , F3G2 and F3G3 comprise glucose and mannose as major components together with smaller amounts of other sugars, including fucose N-acetylglucosamine, xylose and rhamnose, The percentage of galactose is apparently less in F3G2 and F3G3 than in other fractions.
  • RT Reverse transcription
  • PCR polymerase chain reaction
  • RT Reverse transcription
  • PCR polymerase chain reaction
  • the reaction was carried out as follows: 1 ⁇ g of RNA, 1 ⁇ l_ of primer (Oligo(dT) 20 ), and 2 ⁇ l_ of 10 mM dNTP Mix were added to each 0.2 ml_ tube and the total volume was adjusted to 12 ⁇ L with DEPC H 2 O (0.1 % diethylpyrocarbonate-treated H 2 O).
  • the mixture was incubated at 65 0 C for 5 min and immediately chilled on ice. The following was added to each tube as an 8 ⁇ L mixture: 4 ⁇ L of 5 * cDNA buffer, 1 ⁇ L of 0.1 M dithiothreitol (DTT), 1 ⁇ L of RNaseOut (a ribonuclease inhibitor) and 1 ⁇ L of Thermoscript R/T, and 1 ⁇ L of DEPC water.
  • the mixture was incubated at room temperature for 10 min and then 50 0 C for 1 h to allow the first strand of cDNA synthesis. Enzyme activity was terminated by incubating the reactions at 85 0 C for 5 min and the tubes were then placed on ice for
  • Each sample (2 ⁇ L) was added to each reaction tube and the following reagents were added as a 25 ⁇ L mix: 2.5 ⁇ L of 10 x PCR buffer, 2 ⁇ L of 10 mM dNTP Mix, 2.5 ⁇ L of 10 mM each primer (sense and anti-sense), 13 ⁇ L of DEPC H 2 O, and 1 ⁇ L of ProZyme ® (DNA polymerase, from PROtech Technology).
  • reaction tubes were placed in a Strategene PCR Robocycler (Gradient 96) and run under the following condition: one cycle at 94 0 C for 2 min (initial denaturation), then 25 consecutive cycles of 94 0 C for 1 min (denaturation), primer annealing (various temperatures depending on primers, see Table V) for 1 min and 72 0 C for 1 min (primer extension).
  • the reactions were analyzed by gel electrophoresis.
  • the RT-PCR was performed using the specifc primers and the experimental conditions shown in Table V.
  • c WSR water soluble extract of Reshi.
  • Fraction 3 is able to stimulate inflammatory response associated with the expression of IL-1 , IL-6 and TNF- ⁇ , antitumor activity associated with the expression of INF- ⁇ and TNF- ⁇ (see in part. Wang et al 1997) and hematopoiesis associated with the expression of GM-CSF, G-CSF, and M-CSF in view of reports that the anti- apoptotic effect of Reishi Polysaccharide on neutrophilis primarily relies on the expression of GM-CSF (Klein et al 2000).
  • Example 8 Detection of cytokine activity by colorimetric sandwich ELlSA
  • Fresh spleen cells were harvested from BALB/c mice (6-10 weeks old), treated with RBC lysis buffer to destroy red blood cells, and adjusted to 3 x 10 6 cells/mL. Splenocytes were then subject to treatment with either F3, F3G1 , or F3G2 at 100 ⁇ g/mL and incubated in RPMI-1640 medium (GIBCO) containing 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, and 10% FBS at 37 0 C, 5% CO 2 . Supematants were collected at indicated time intervals by centrifugation at 30Og for 10 min. In vitro lFN- ⁇ and GM-CSF activity was determined using the Quantokine ® Murine ELISA kit (R&D System Inc., Minneapolis, USA) following manufacturer's instructions.
  • sample supematants 50 ⁇ L and 50 ⁇ L of sample supematants, standard and control, were gently mixed and loaded into individual wells. After 2 h of incubation at room temperature, each well was aspirated, washed five times with 400 ⁇ L of Wash Buffer, loaded with 100 ⁇ L of secondary antibody solution (conjugated with horseradish peroxidase) at room temperature for 2 h.
  • the concentration of cytokine released was determined by plotting the sample reading against the standard curve. To rule out possible LPS contamination, TNF- ⁇ expression upon administration of LPS at various concentrations, was also measured.
  • GM-CSF GM-CSF
  • Fig. 17 IFN-Y
  • Figs. 18 and 19 TNF- ⁇ .
  • Similar enhancement was observed when the ELISA assay was carried out for the IFN- ⁇ expression (Fig. 17).
  • the treatment with F3G2 and F3 was able to stimulate the spleen cells to generate 143 and 8.6 pg/mL IFN- ⁇ , respectively, after the 48-h incubation.
  • the respective dose dependent profiles appear to be dissimilar (Figs. 18 and 19): while LPS reached a plateau at the concentration of 10 ⁇ g/mL, F3G2 profile appears as a sigmoidal curve.
  • F3G2 shows to be more active than LPS at dosages higher than 100 ⁇ g/mL, a result that rules out possibility of LPS contamination and supports the conclusion that the molecular mechanism activated by the two molecules are different.
  • Lucidum PS extract prepared via alkaline extraction with 0.1 N of NaOH, followed by neutralization and ethanol precipitation, was obtained from Pharmanex (CA).
  • the crude G. Lucidum extract (100 g) was dissolved in 3 L of double-distilled H 2 O and stirred at 4°C for 24 h. The solution was centrifuged (16,000 g) at 4°C for 1 h, and the supernatant was concentrated at 35°C. The slurry product was then lyophilized to obtain 70 g of water-soluble dark brown G. Lucidum extract.
  • the extract (2.5 g) was fractionated on Sephacryl S-500 column (95 2.6 cm) with 0.1 N of Tris buffer (pH 7.0) as the eluent.
  • the flow rate was set at 0.6 ml/min, and fractions were collected with 7.5 ml per tube. Five fractions were collected, and each was dialyzed to remove excessive salts and lyophilized to give fractions 1-5; each fraction was characterized, as described in Example 1.
  • Lucidum materials and PS extracts were prepared, from growth to harvest, as GMP grade from Pharmanex and the possible bacterial contamination was carefully monitored to meet the Food and Drug Administration standard.
  • the reagents and utensils for preparation of F3 were either endotoxin-free grade or washed with PBS containing 50 ⁇ g/ml polymyxin B (PMB), then rinsed with PBS.
  • F3 contained ⁇ 1 ng of LPS/25 ⁇ g, as measured by LAL assay (Sigma-Aldrich).
  • certain reagents were routinely checked by LAL for examination of LPS contamination. An additional procedure was performed as described in Wang et al 2002 herein incorporated by reference in its entirety.
  • a modified version of the procedure described in Want et al. 2002 comprises, direct centrifugation, isolated polysaccharide from water soluble Reishi sample which showed components as well as F3.
  • This portion of polysaccharide shown familiar HPLC profile with F3 and its bio-function assay was analyzed, such as effect of proliferation and cytokines release to mice splenocyte, as well as previously F3 function
  • TLR4 functions as a receptor for F3 and mediates F3-induced transducing signals
  • HeNC2 peripheral macrophages from wild-type C3H/HeN mice with functional TLR4
  • GG2EE peripheral macrophages from C3H/HeJ mice lacking functional TLR4
  • HeNC2 and GG2EE were treated for 6 h with F3 as prepared in Example 9 (25 ⁇ g/ml) and LPS (1 ⁇ g/ml), respectively.
  • Whole cell lysates were analyzed by Western blot with anti-IL-1 antiserum, as previously described (Hsu et al 2001 ; Hsu et al 2002)
  • Fig. 20 show that HeNC2 exhibited pro-IL-1 production upon F3 stimulation (25 ⁇ g/ml) (Fig. 20, sample 3), whereas no detectable pro-IL-1 was found in F3-treated GG2EE (Fig. 20, sample 3 vs. 6).
  • the human primary macrophages were in particular pre-incubated with the mouse anti-human TLR4 mAb (HTA125) or CR3 mAb (10 ⁇ g/ml) for 30 min, followed by treatment with F3 (25 ⁇ g/ml) and LPS (1 ⁇ g/ml), respectively, for 6 h.
  • Western blot analyses of pro-IL-1 were conducted, as described previously (Hsu et al 2001 ; Hsu et al 2002).
  • Results are shown in Fig. 21.
  • HTA125 mAb inhibited LPS-induced TLR4- mediated induction of pro-IL-1 expression (Fig. 21 , sample 3) and F3-mediated pro-IL-1 expression (Fig. 21 , sample 5).
  • CR3 mAb failed to block F3-induced pro-IL-1 production (Fig. 21, sample 4 vs. 7) indicating that F3 is not recognized by CR3.
  • TLR4 and not CR3 is a receptor for F3.
  • Additional experiments were also carried out to test the effect of F3 administration on the IL-1 R associated kinase (IRAK), a serine/threonine kinase, included in the TLR signaling complex together with myeloid differentiation factor 88 (MyD88), an adaptor protein.
  • IRAK IL-1 R associated kinase
  • MyD88 myeloid differentiation factor 88
  • IRAK Upon phosphorylation (auto- or cross- phosphorylation), IRAK loses its affinity to the TLR signaling complex, is rapidly released from the coplex and degraded by proteosomes.
  • Macrophages were cultured with F3 or LPS for time intervals ranging from 0 to 120 minutes, the cells lysed and total cell lysates were used for western blot analysis, using an IRAK antibody.
  • the results, illustrated in Fig. 60, show that both LPS and F3 induce rapid degradation of IRAK protein at a similar rate. Those results further support the conclusion that TLR4 is the F3 receptor.
  • F3 is LPS free and different from LPS in ROS production, pro- IL-1 stimulation in presence of PMB, and protein tyrosine phosphorylation (PTP)
  • CSH 2 DCFDA 5(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester
  • J774A.1 cells were preincubated with CM-H 2 DCFDA (2 ⁇ M) for 30 min, followed by substitution with medium containing F3 (25 ⁇ g/ml) or LPS (1 ⁇ g/ml) for additional incubation for the indicated times.
  • the relative fluorescence intensity of fluorophore CM-H 2 DCF was detected, as previously described (Hsu et al. 2002). The results, illustrated in Figs.
  • J774A.1 cells were pre-incubated with or without 10 ⁇ g/ml PMB for 30 min, followed by treatment with F3 (25-200 ⁇ g/ml) or LPS (0.01-1 ⁇ g/ml) for 6 h.
  • F3 25-200 ⁇ g/ml
  • LPS (0.01-1 ⁇ g/ml
  • Fig. 25 show that incubation of human macrophages with F3 induces the appearance of many phosphotyrosyl proteins, compared with phosphotyrosyl proteins appearing upon treatment with LPS or medium alone.
  • some tyrosine-phosphorylated proteins of molecular mass at 38, 42-44, and -52 kDa were identified (Fig. 25, sample 1 vs. 2, 3, 4, and 5) and immunoreacted with anti-p38 IgG (38 kDa), anti-ERK IgG (42-44 kDa), and anti-JNK IgG (-52 kDa), respectively (data not shown).
  • Example 12 F3 up-requlates IL-1 secretion, pro-IL-1 protein, and message expression, and stimulates the activity of IL-1 -converting enzyme (ICE or caspase 1) in human macrophages and J774A.1 cells
  • ELISA experiments were performed as previously described (Hsu et al 2001 ; Hsu et al 2002) to quantify mature IL-1 secretion in the conditioned medium of J774A.1 cells.
  • -60, 140, and 200 pg/ml of secreted IL-1 protein was detected in the conditioned medium of 6-, 12-, and 24-h F3-treated cells, respectively (Fig. 26).
  • the production of IL-1 by treated cells was detected in comparison with the one by control cells (production below a detectable level), and continued to increase to 300 pg/ml after 36 h.
  • the IL-1 detected upon F3 induction relative to the IL-1 detected upon LPS induction was -40 pg/ml (6 h), 130 pg/ml (12 h), and 160 pg/ml (24 h), respectively, and then decreased after 36 h.
  • cells were treated with F3 (12.5 and 25 ⁇ g/ml) or LPS (0.5 and 1 ⁇ g/ml) for 6 h; whole cell lysates treated with different amounts of F3 and/or LPS were then analyzed by Western blot with anti-IL-1 antiserum, as described in (Hsu et al 2001 ; Hsu et al 2002).
  • LPS on pro-IL-1 induction was found in a sample from cells treated with F3 (12.5 ug) plus LPS (0.5 ug)(sample 4 of Fig. 27) and a sample from cells treated with
  • J774A.1 cells and human macrophages was tested during a time course from 0 to 24 hours.
  • Cells were treated with F3 for various times and concentrations, as indicated in Figs. 28 and 29; whole cell lysates were then analyzed by Western blot with anti-IL-1 antiserum, as described in (Hsu et al 2001 ; Hsu et al 2002).
  • the effects of F3 on IL-1 secretion and pro-IL-1 expression in J774A.1 cells and in human macrophages are shown in Fig. 28 and 29, which illustrate time course (Fig. 28) and dose response (Fig. 29) of pro-IL-1 expression in F3- treated cells.
  • Fig. 28 and 29 illustrate time course (Fig. 28) and dose response (Fig. 29) of pro-IL-1 expression in F3- treated cells. The results, illustrated in Fig.
  • IL-1 secretion via lnterleukin Converting Enzyme have been reported in various cells, including macrophages with LPS (Hsu et al 2002). Because incubation of macrophages with F3 induced the expression of pro-IL-1 and increased IL-1 secretion in a time-dependent fashion (Fig. 28 vs. 26), F3 induced ICE activity during IL-1 secretion was tested.
  • Cells were treated with F3 for various times or with LPS for 18 h; cell extracts (90 ⁇ g of protein) were then incubated in the presence of the fluorescence ICE substrate Ac-YVAD-AMC (50 ⁇ M) for 1 h at 3O 0 C.
  • the ICE activity was measured fluorometrically with excitation at 360 nm and emission at
  • F3 increased ICE activity up to 1.5-fold between 6 and 12 h, and gradually reached to 2-fold at 24 h, compared with the control cells or the LPS-treated cells for 18 h.
  • IL-1 secretion is consistent with the sequential synthesis of pro-IL-1 mRNA and pro-IL-1 as well as with induced ICE activity after F3 stimulation (see Fig. 31 ).
  • Example 13 F3 activates MAPK phosphorylation and activity in J774A.1 ceils and human macrophages
  • the activated ERK and p38 were immunoprecipitated from cell lysates using specific Abs.
  • Recombinant Elk-1 fusion protein and ATF-2 fusion protein were used as substrates for ERK and p38, respectively.
  • F3-induced ERK and p38 activities were monitored by phosphorylation of substrate. These were measured by quantitative immunoblotting with phospho-Elk-1 (Ser 383 ) Ab and phospho-ATF-2 (Thr 71 ) Ab, respectively. Histogram shown in Figs.
  • 35 and 36 represents quantification by Phosphorlmager of F3-stimulated phospho-ERK1/2, phospho-JNK1/2, and phospho-p38 in J774A.1 cell sample using ImageQuaNT software from Amersham Pharmacia Biotech (Sunnyvale, CA).
  • the F3-stimulated phospho-ERK1/2, phospho-JNK1/2, and phospho-p38 activities and the F3-stimulated ERK1/2 and p38 activity in J774A.1 cell sample was quantified by Phosphorlmage using ImageQuaNT software from Amersham Pharmacia Biotech
  • ERK The activated, Thr ⁇ /Tyr ⁇ -phosphorylated form of ERK was detected upon F3 administration to J774A.1 cells.
  • phosphorylated ERK Extracellular signal Regulated Kinase
  • J774A.1 cells in -10 min reached the maximum level in 20 when the concentration became 3-fold higher compared to the initially detected concentration.
  • ERK phosphorylation gradually returned to the basal level and was detected again in -120 min (Fig. 32 and 35).
  • Further studies on human macrophages showed a similar trend the in the phosphorylation of ERK (data not shown).
  • F3 simulation of ERK activity was demonstrated by the phosphorylation of Elk, a downstream substrate of ERK (Fig. 32 and 36).
  • J774A.1 cells were exposed to concentrations of inhibitors, as indicated, followed by incubation with F3.
  • J774A.1 cells were treated with Ro- 31-8220, PD98059, SP600125, and SB203580 on F3-induced pro-IL-1 expression.
  • Cells were preincubated with various concentrations of inhibitor, as indicated in Figs. 37 to 40, for 1 h, followed by F3 stimulation (25 ⁇ g/ml) for additional 6 h.
  • Example 15 effect of lv294002 (an inhibitor of pi3k) on f3-induced ink and p38 activity as well as on F3 regulation of PRO-IL1 and 11-1 secretion
  • LY294002 a specific inhibitor of PI3K was used.
  • LY294002 was administered to J774A.1 cells to examine the possible involvement of PI3K in F3-mediated activation of JNK and p38.
  • J774A.1 cells were pretreated with various concentrations of LY294002 (5, 25, 50, and 100 ⁇ M) for 1 h before stimulation with F3 (25 ⁇ g/ml) for additional 20 min, followed by measuring the phosphorylation of JNK and p38, as described previously.
  • FIG. 41 shows that in the absence of LY294002, F3 induced the phosphorylation of JNK and p38 by ⁇ 13-fold (Fig. 41 , sample 2 vs. 1 ) and 10-fold (Fig. 41 , sample 2 vs. 1 ), respectively, compared with the control (Fig. 41 , sample 1 ).
  • Fig. 41 samples 3, 4, 5, and 6 vs. 2
  • PI3K was less or not associated with F3 activation of JNK and p38.
  • J774A.1 cells were pretreated with LY294002 (50 ⁇ M) for 1 h, followed by incubation with F3 (25 ⁇ g/ml) for additional 6 h, and Western blot analyses of pro-IL-1 expression were conducted, as previously described (Hsu et al 2001 ; Hsu et al 2002). Similar results were obtained in three separate experiments.
  • LY294002 on F3-induced IL-1 secretion was detected by ELISA.
  • Cells preincubated with LY294002 before F3 treatment showed a significant reduction in the secreted IL-1 to 5 pg/ml (6 h), 14 pg/ml (12 h), and 18 pg/ml (24 h), respectively, in the conditioned medium (Fig. 41 ), which was much lower than the corresponding F3-treated control cells.
  • L human umbilical cord blood
  • MNC mononuclear cells
  • CD1 a, CD14, CD19, CD26, CD56 CD83 which are markers for specific cell differentiation.
  • CD19 is a B cell specific antigen, is a critical signal transduction molecule that regulates B lymphocyte development, activation, and differentiation (Carter et al 2002);
  • CD14 is a monocyte/macrophage differentiation marker (Dobrovolskaja and Vogel 2002);
  • CD26 is a cell surface protease, expressed on many cells of the immune system including some CD4 + T-cells and macrophage (Morimoto and Schlossman 1998); anti-human CD56 and CD16 antibodies are both used for identification of natural killer (NK) cells and their subpopulations, according to the different expression of the surface antigens CD16 and CD56(Cooper et al 2001);
  • CD83 and CD1a are expressed on dendritic cells while anti-CD83 and anti-CD1 a antibodies can serve as useful markers for human dendritic cells phenotypic characterization (Foley
  • Human umbilical cord blood from six healthy volunteers was drawn into EDTA-coated tubes. The blood was collected right after the full-term baby was delivered and before the placenta separated from the uterus. Using aseptic procedures, an 18-gauge needle was inserted into the umbilical vein and umbilical cord blood drawn for tests. Samples were stored at room temperature and processed within 24 h after collection. The umbilical cord blood (50-100 ml_) was processed using density gradient centrifugation with Ficoll-Paque (density 1.077; Pharmacia Biotech; Uppsala, Sweden).
  • the buffy coat interface was retrieved and washed with Dulbecco's phosphate buffered saline ([PBS] pH 7.4) and EDTA (0.2 mM). It was re- suspended in a complete culture medium, consisting of RPMI-1640, 2 mM L- glutamine, 100 IU/mL penicillin, 100 ⁇ g/mL streptomycin (Gibco BRL), and was then supplemented with 20% fetal bovine serum (FBS). Mononuclear cells isolated through these procedures were prepared at a final concentration of 10 6 cells/mL
  • Mononuclear cells isolated from the six umbilical cord blood specimens were placed in six T75 culture flasks at 5 * 10 5 cells/mL density in preparation for the F3 treatment. After seeding of cells, the flasks were maintained in a 37 0 C to 5% CO 2 incubator for 1 h to equilibrate before 100 ⁇ g/mL of G. Lucidum F3 extracts were added to each culture. The F3 fraction was dissolved in PBS for all experiments. Control cultures were added with an equal volume of PBS without F3, while positive controls were treated with 100 ⁇ g/mL LPS (Sigma) from a Gram- negative cell wall.
  • Cells were cultured for 7 days after treatment. To proceed for flow cytometry, cells (1-2 x 10 6 ) were pelleted and re-suspended in 2 mL of staining buffer (0.2 mM EDTA, 2% FBS in phosphate buffered saline [PBS]). Staining buffer (100 ⁇ L) containing 10 ⁇ L of fluorescence-conjugated antibody was added to the cell suspension for labeling. After incubation at 4 0 C for 40 min, all samples were then centrifuged at 1500 rpm for 5 min, followed by washing of the pellets twice with washing buffer (0.2 mM EDTA, 2% FBS in phosphate buffered saline [PBS]).
  • staining buffer 0.2 mM EDTA, 2% FBS in phosphate buffered saline [PBS]
  • Cells were therefore treated with F3 at different concentrations and with LPS and then to monoclonal antibodies specific for surface antigens to identify the different cell types. All monoclonal antibodies to surface antigens, including CD45, CD3, CD16, CD19, CD56, CD83, and CD1a, were obtained from Coulter Immunotech, USA.
  • Flow cytometry was performed with a FACSclibur cytometer (Becton Dickinson). The instrument was set for two-color analysis using FACScomp software and was calibrated using Calibrite beads (Beckton Dickinson) with a threshold of 200 on FSC to exclude debris. Data were collected in list mode and analyses were performed using CellQuest software version 3.1f (Becton
  • CD34 + hematopoietic stem cell 78.0 ⁇ 12% 151.0 ⁇ 14% 151.0 ⁇ 15%
  • CD14 + CD26 + monocyte/macrophage, CD83 + CDIa + dendritic cells, and CD16 + CD56 + NK-cells increased as a result of UCB exposure of mononuclear cells from human umbilical cord blood (UCB), to F3 at about 10- 100 ⁇ g/ml for 7 days.
  • Cell numbers were determined using light microscopy, based on the ability of living cells to exclude trypan blue. Cell proliferation was assessed by reduced activity on sodium (2,3)-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H- tetrazolium-5-carboxanilide, inner salt (XTT) (Rohem et al 1991 ; Scudiero et al 1988). Briefly, 100 ⁇ L of 2 x 10 5 cells/mL were incubated with different concentrations of F3 for 48 h. An XTT solution containing phenazine methosulfate was added to a final concentration of 0.2 mg/mL and 25 mM, respectively, for 4 h. Absorbance was measured with a spectrophotometer, using test and reference wavelengths of 450 and 650 nm, respectively. Each experiment was performed in triplicate and repeated at least three times. Results were expressed as the mean ⁇ SEM.
  • Example 17 Lysis of K562 cells after enrichment of CD56* NK-cells
  • CD56 + natural-killer cells isolated from UCB mononuclear cells were enriched by a positive magnetic-bead cell separation method (MACS, Miltenyi Biotec). MNC from the buffy coat of human umbilical cord blood were isolated by using Ficoll-Paque ® as mentioned above, and passed cells through 30 ⁇ m nylon mesh (Millipore) to remove clumps. (The filter should be rinsed before use.) Filtered cells were washed twice with buffer (PBS containing 0.1 % sodium azide, 1 % human serum albumin, and 0.15% sodium citrate).
  • buffer PBS containing 0.1 % sodium azide, 1 % human serum albumin, and 0.15% sodium citrate
  • the cell pellets were suspended in 500 ⁇ l_ of this buffer and 200 ⁇ L of FcR Blocking Reagent (Miltenyi Biotec) and incubated for 15 min on ice to block FcR. Then, 200 ⁇ L of CD56 + microbeads per 10 8 total cells were added, followed by incubation for an additional 30 min on ice, and then washed twice with the buffer. The cells were re-suspended in 1 ml_ of the buffer.
  • FcR Blocking Reagent Miltenyi Biotec
  • the magnetically labeled cells in 1 ml_ of the buffer were applied to two MACSy RS 1 separation columns (Miltenyi Biotec) that had been equilibrated with the buffer in the magnetic field of the Vario MACSy separator (Miltenyi Biotec) (Gritzapis et al 2002).
  • the negative cells were washed off the column with 2 ml_ of the buffer.
  • Retained cells were eluted from the column outside the magnetic field by pipetting 2 ml_ of the buffer onto the column and using the plunger supplied with the column.
  • Aliquots of the sorted cells were stained by PC5-labeled anti-CD56 + monoclonal antibody (Coulter Immunotech) to analyze the purity of CD56 + NK- cells.
  • the purity of the isolated NK-cells was determined by flow cytometry analysis and reached up to 95%.
  • target cells K562 (CCL-243, ATCC), a human erythroleukemia cell line, was used as an NK-sensitive target for the cytotoxicity assays.
  • NK-cells Different concentrations of activated effector cells, that is, F3-treated CD56 + NK-cells, and target cells, that is, K562, were co-cultured in six-well plates (Falcon) in triplicate.
  • the effector to target cell ⁇ E:T) ratios were 5:1 , 20:1 , and 80:1 , respectively.
  • the cytotoxic activity of NK-cells was measured using Alamar Blue (Alamar BioSciences, Sacramento, CA), which is a colorimetric indicator that changes color upon reduction when a membrane potential across a cell occurs (Zhang et al. 2000).
  • Figs. 48 to 51 The results are shown in Figs. 48 to 51 , wherein the data reported are the mean ⁇ SD of three independent experiments.
  • the highest level of cytotoxicity was noted at an E/T ratio of 20:1 when the effector cells were pre-incubated with 100 ⁇ g/ml_ F3 concentration.
  • NK- cytotoxicity increased by 31.7% (P ⁇ 0.01 ) and 20.1 % (P ⁇ 0.05) after pre- treatment with 100 and 50 ⁇ g/mL of F3, respectively, when compared to the untreated controls (Figs. 48 and 50).
  • the cytotoxicity at an E/T ratio of 5:1 was not significant, compared to the controls.
  • UCB MNCs were collected from six volunteers. These MNCs were cultured for 7 days after treatment with 100 ⁇ g/mL of F3. NK-cells were then harvested and tested for cell surface markers (CD56 and CD3) using two-color flow cytometric analysis. Control groups were treated with PBS only.
  • the antigenic peptides on MHC molecules expressed by antigen presenting cells interact with T cells, and promote clonal proliferation of antigen- specific T cells, which occupy central roles between the innate and adaptive immune responses.
  • the interactions among co-stimulatory molecules (including CD80 and CD86) and CD28/CTLA-4 are crucial in the regulation of T cell activation and tolerance. This phenomenon prompted the inventors to investigate the effect of Reishi Fraction 3 on MHC class I (HLA-DA, DB, and DC), MHC class Il (HLA-DR, DP, and DQ) and CD80/CD86 expression in antigen presenting cells, such as human primary monocytes, macrophages and dendritic cells. Human primary monocytes and dendritic cells were isolated from blood of healthy persons obtained from Taiwan Blood Center (Taipei, Taiwan).
  • Monocytes or dendritic cells incubated with Reishi exhibit an increase in surface MHC class Il expression, yet no change in the expression of MHC class I.
  • Reishi increases surface expression of the co-stimulatory molecules CD80 and CD86 on monocytes and dendritic cells.
  • Example 20 Reishi F3 is associated with induction of Blimp-1 in purified mouse splenic B cells
  • Blimp-1 B lymphocyte-induced maturation protein-1 . plays a crucial role during plasmacytic differentiation. Ectopic expression of Blimp-1 is sufficient to induce plasma cell formation and immunoglobulin secretion from mature B cell in mouse B cells. B cell specific prdmi, gene encodes for Blimp-1 , deficient mice revealed a severe defect on plasma cell differentiation.
  • Blimp-1 protein is induced in mouse splenic B cell culture after treatment with Reishi F3 (Fig. 56) and that Blimp-1 mRNA is highly induced when mouse splenic B cells are stimulated with Reishi F3 (Fig. 55).
  • the effects of F3 administration on purified human peripheral B cells were also tested. To this extent, purified human peripheral B cells from different donors (donor #1 ) (donor #2) and (donor #3) were treated with Reishi F3 for 6 days and then subjected to RNA isolation and RT reaction using primer and probe sets to Blimp-1 and normalized to PPIA's expression level.
  • Reishi F3 or F3G2 fractions caused up-regulation of Blimp-1 mRNA and protein after three days of treatment in mouse splenic B cell culture.
  • RT-QPCR and western blot was used to determine the induction of Blimp-1 mRNA and protein.
  • Dried Reishi F3 powder was dissolved in H 2 O before addition to B cell culture.
  • Cells treated with Reishi for three days, were subject to RNA isolation.
  • Real-time quantitative RT-PCR was used to monitor the changes of folds of expression of Blimp-1 mRNA.
  • the induction of Blimp-1 mRNA was normalized to the endogenous L32 mRNA level.
  • Blimp-1 is a molecular marker associated with Reishi stimulation in mouse splenic B cells and human peripheral B cells.
  • Reishi F3 induces the expression of Blimp-1 mRNA in human dendritic cells.
  • Human dendritic cells from healthy donors were treated with Reishi F3 and the levels of Blimp-1 mRNA were determined.
  • isolated human dendritic cells were treated with 10 ⁇ g/ml Reishi F3 and subjected to RT-QPCR at day 7 QPCR using primer and probe sets to Blimp-1 and normalized to PPIA's expression level, with untreated human dendritic cells were used as a control.
  • Example 22 PS Inhibit macrophage (RAW 264.7) expression of iNOS induced by LPS
  • Macrophages play an important role in the early immune response of mammals.
  • the immune functions of macrophage in the early immune response include production of Nitric Oxide (NO) which is generated by inducible nitric oxide synthase, such as iNOS, eNOS, Type Il NOS, upon exposure of the macrophage to certain cytokines, such as interferon- ⁇ . NO production by the macrophage is mediated through a series of signal transducers including the Janus Kinase family (JAK) and signal transducers and activators of transcription (STAT) proteins.
  • NO Nitric Oxide
  • iNOS inducible nitric oxide synthase
  • eNOS eNOS
  • Type Il NOS cytokines
  • Table IX shows the characteristics of different nitric oxide synthase isozymes including expression, rate limiting, product quantity, chromosomal location, enzyme location and molecular weight (Mw).
  • Oxidative Stress Reactive Nitrogen Species e.g. Peroxynitrite
  • PAF platelet-activating factor
  • PG prostaglandin
  • IL-1-ra interleukin-1 -receptor antagonist
  • TNF tumor necrosis factor
  • mAb monoclonal antibody
  • SR soluble receptor
  • iNOS is produced in LPS-induced sepsis, which includes a early phase (0-1.5 hr) including the direct release of vasorelaxant mediators, and a delayed phase (1.5-24 hr) including the expression of isoforms of iNOS as well as the other inducible enzymes PLA 2 -II (type Il phospholipase A 2 ) and COX- Il (cyclooxygenase-ll).
  • PLA 2 -II type Il phospholipase A 2
  • COX- Il cyclooxygenase-ll
  • RAW 264.7 (5x10 5 cells/well) were cultivated in DMEM 10% FCS for 24 hours, and in DMEM 0% FCS for additional 4 hours.
  • the culture medium was then replaced with DMEM 2%FCS and the cells were treated with 10-1000 ⁇ g/mL F3 with or without LPS at 3, 5, and 10 ng/mL.
  • Measurement of NO 2 concentration in the culture medium and measurement of cell viability were performed after 24 hours.
  • the NO 2 " concentration was measured by Nitrite Assay in vitro, according to the procedure schematically represented in Fig. 62.
  • the supernatant from the cell culture was mixed with Greiss reagents in proportion 1 :1 for 10 minutes, the resulting light absorbance in OD was measured at 550 nm.
  • Fig. 65 show that the F3 (herein also GL(PS)_Wu or Wu) treatment with or without LPS does not affect the viability of the cells in the culture
  • a fucose-containing glycoprotein fraction obtainable from an extract of Ganoderma Lucidum Reishi, a method to mediate immunomodulating events associated with IL-1 gene expression, a method to stimulate the expression of an inflammatory cytokine, a method to modulate protein kinase pathways associated with inflammatory cytokine lnterleukin 1 , a method to induce a TLR mediated event; a method to modulate differentiation of a mononuclear cell, a method to enhance cytotoxicity of an NK cell against an NK- sensitive tumor cell, a method to activate the expression of cytokines, a method to induce Blimp-1 expression in a mouse splenic B cell or human B cell, human mature splenocytes or dendritic cell, and a method to inhibit LPS induced nitric oxide production in macrophages.

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WO2006044616A3 (en) 2006-08-24
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EP1804584A4 (de) 2009-08-05

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